Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "COT drives resistance to RAF inhibition through MAPK pathway reactivation" by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Introduction

Activation of the canonical mitogen activated protein kinase (MAPK) pathway occurs in response to the binding of growth factors, hormones, or neurotransmitters to receptor tyrosine kinase receptors located at the cell surface (Dhomen and Marais, 2009; Lopez-Bergami et al., 2008). In untransformed cells, receptor ligation induces the sequential activation of the small GTPase RAS, followed by RAF, MEK and ERK, which relays proliferative signals generated at the cell periphery into the nucleus to control cellular survival, differentiation and growth (Inamdar et al., 2010; Panka et al., 2006). Not surprisingly, dysregulation of MAPK signaling is common in many human cancers including melanoma. Mutations in the RAF and RAS genes (Davies et al., 2002; Mercer and Pritchard, 2003) that trigger constitutive activation of the MAPK pathway can result in uncontrolled cell proliferation, invasion, metastasis, survival and angiogenesis (Panka et al., 2006; Sharma et al., 2006; Smalley et al., 2006; Smalley and Herlyn, 2006).

BRAF is one of three members of the RAF family, which includes ARAF, BRAF, and CRAF (or RAF-1) (Dhomen and Marais, 2009). In melanoma, BRAF represents the most commonly mutated gene in the MAPK signaling cascade where 90% of tumors carry a valine to glutamic acid transition at codon 600 (V600E) that renders BRAF constitutively active and hyperactivates the MAPK cascade (Davies et al., 2002; Dhomen and Marais, 2009; Michaloglou et al., 2008). While preclinical and clinical studies have shown that targeting BRAF (V600E) melanomas with the use of RAF-selective inhibitors results in initial tumor regression (Fedorenko et al., 2015; Flaherty et al., 2010; Shtivelman et al., 2014), responses to RAF inhibitors are transient, with acquired resistance triggering disease progression (Shtivelman et al., 2014). Although progress has been made in the development of drugs that target RAF, the clinical outcome regarding long-term usage and the mechanisms of acquired resistance warrants further evaluation. In their study, Johannessen and colleagues sought to identify kinases involved in mediating resistance to the RAF kinase inhibitor PLX4720 (Johannessen et al., 2010).

Using a kinase open reading frame (ORF) collection and a high throughput screening methodology, Johannessen and colleagues identified MAP3K8 (the gene encoding cancer osaka thyroid (COT)/TPL2), as a driver of resistance to BRAF inhibition with PLX4720 (Johannessen et al., 2010). Johannessen and colleagues first examined basal MAP3K8 expression in multiple cell lines harboring the V600E mutation (Johannessen et al., 2010). As shown in Figure 3B and reported by others, RPMI-7951 and OUMS-23 cells were found to express high intrinsic levels of MAP3K8 compared to A375 cells where MAP3K8 was undetectable (Johannessen et al., 2010; Paraiso et al., 2012). RPMI-7951 and OUMS-23 cells also exhibited robust, undiminished ERK and MEK activity concomitant with reduced growth sensitivity in the presence of PLX4720 (Figure 3D–E; Johannessen et al., 2010). This is supported by additional findings demonstrating that RPMI-7981 cells treated with the closely related BRAF inhibitor PLX4032/vemurafenib (a successor of PLX4720) also remain refractory to inhibitor treatment as assessed by annexin V staining (Paraiso et al., 2012) and MTS assay (Park et al., 2013). However, others have reported RPMI-7981 cells as exhibiting modest sensitivity to PLX4720 (Schayowitz et al., 2012). In the latter case, ERK activity was reduced by 50% after incubation with inhibitor, although these differences in sensitivity may reflect the significantly shorter time course and experimental design used by Park and colleagues. Finally, in Figure 3I, Johannessen and colleagues determined that MAP3K8 kinase activity is required to regulate MEK/ERK activation in RPMI-7951 cells, findings that further confirm MAP3K8 is an essential upstream activator of the MEK-ERK signaling cascade (George and Salmeron, 2009; Johannessen et al., 2010). The key experiments outlined in Figures 3B,D,E, and 3I will be replicated in protocols 1, 2, 3, and 4.

Resistance to targeted agents, such as BRAF inhibitors, is a frequent cause of therapy failure, as noted above. Importantly, chronic BRAF inhibition can lead to cross-resistance to several BRAF-selective inhibitors, indicating that resistance is not likely to be overcome by switching to a new RAF inhibitor (Corcoran et al., 2010; Villanueva et al., 2011). It has been suggested previously that combination treatment with MEK and BRAF inhibitors may be useful in preventing the emergence of resistance or in overcoming resistance to single agent therapies targeting either molecule alone (Corcoran et al., 2010). To examine whether the combined use of RAF and MEK inhibitors bypass MAP3K8-driven resistance, Johannessen and colleagues ectopically expressed MAP3K8 in A375 melanoma cells before treatment with BRAF inhibitor (PLX4720) alone or in combination with the MEK inhibitors CI-1040 or AZD6244. As shown in Figures 4E and 4F, both viability and ERK activation was dramatically reduced in MAP3K8-expressing cells treated with either of the combination therapies, similar to cells ectopically expressing MEK1, which remained sensitive to PLX4720 (Johannessen et al., 2010). Similar results were obtained in RPMI-7951 cells expressing high basal levels of MAP3K8 treated with PLX4032 and a second MEK inhibitor AS703026 (Park et al., 2013). Interestingly, overexpression of constitutively active MEK (MEK1DD) resulted in increased sensitivity to BRAF inhibition combined with AZD6244, but not CI-1040 (Johannessen et al., 2010). These findings confirm that MAP3K8 is able to reactivate MAPK signaling despite BRAF inhibition and that targeting RAF and MEK in combination may be an effective anti-melanoma treatment strategy. These experiments will be replicated in Protocols 5 and 6.

Materials and methods

Protocol 1: MAPK pathway analysis in cells expressing elevated MAP3K8

This experiment assesses the effect the RAF inhibitor, PLX4720, has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiments reported in Figures 3B and 3E.

Sampling

Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

See Power Calculations section for details.

Experiment has 3 cohorts:

Cohort 1: A375 cells

Cohort 2: RPMI-7951 cells

Cohort 3: OUMS-23 cells

Each cohort has four conditions:

Vehicle (DMSO)

10 µM PLX4720

1 µM PLX4720

0.1 µM PLX4720

Each condition will be probed with the following antibodies:

pERK1/2 (T202/Y204)

pERK1/2

pMEK1/2 (S217/221)

MEK1/2

MAP3K8

Actin

Materials and reagents

Procedure

Note:

A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates and incubate for 24–36 hr to achieve log phase growth.

24–36 hr after seeding treat cells with 0.1, 1, and 10 µM PLX4720 or DMSO. Incubate for 24 hr.

Add drug directly to each well using a 1000X stock (in DMSO).

Final DMSO concentration kept to 0.1%.

Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II.

Add ~100–200 µl 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 µg/µl.

b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed (table-top microfuge) at 4˚C.

Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C.

Separate 35–50 μg of protein per lane on a 10% Tris/Glycine gel with protein ladder following replicating lab’s standard protocol.

Samples run per gel:

Protein molecular weight marker

Vehicle (DMSO) treated A375 cells

10 µM PLX4720 treated A375 cells

1 µM PLX4720 treated A375 cells

0.1 µM PLX4720 treated A375 cells

Vehicle (DMSO) treated RPMI-7951 cells

10 µM PLX4720 treated RPMI-7951 cells

1 µM PLX4720 treated RPMI-7951 cells

0.1 µM PLX4720 treated RPMI-7951 cells

Vehicle (DMSO) treated OUMS-23 cells

10 µM PLX4720 treated OUMS-23 cells

1 µM PLX4720 treated OUMS-23 cells

0.1 µM PLX4720 treated OUMS-23 cells

Wet transfer with supplied wet-transfer cassette apparatus to immobilon P following replicating lab’s standard protocol.

Original transfer protocol was for 120min at 30–35 V at 4˚C.

After transfer, block non-specific binding and immunoblot membrane with the following primary antibodies for 18 at 4˚C following manufacturer recommendations:

mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa

mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa

rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa

rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa

rabbit anti- MAP3K8; use at 1:500 dilution; 52, 58 kDa

mouse anti-ß-Actin; use at 1:100 - 1:1000 dilution; 43 kDa

Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions.

Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions.

Analyze bands with image analysis software and normalize to loading controls.

pERK1/2 (T202/Y204) normalized to MEK1/2 (total).

pMEK1/2 (S217/221) normalized to ERK1/2 (total).

MAP3K8 normalized to Actin.

Repeat steps 1–9 independently three additional times.

Data to be collected:

Full image western blot films of all immunoblots including ladder. (Compare to Figures 3B and 3E)

Raw data of band analysis and normalized bands for each sample.

Statistical Analysis of the Replication Data:

Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using the Bonferroni correction:

Planned contrast of normalized pERK1/2 levels from A375 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Planned contrast of normalized pERK1/2 levels from RPMI-7951 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Planned contrast of normalized pERK1/2 levels from OUMS-23 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Planned contrast of normalized pMEK1/2 levels from A375 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Planned contrast of normalized pMEK1/2 levels from RPMI-7951 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Planned contrast of normalized pMEK1/2 levels from OUMS-23 cells treated with vehicle compared to cells treated with PLX4720 (all doses).

Meta-analysis of original and replication attempt effect sizes:

The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

Known differences from the original study

The replication will not include the other BRAF (V600E) cell lines reported in the original paper. The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. The western blots will use Actin, instead of Vinculin, which was reported in Figure 3B. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 2: Determine the range of detection of the replicating lab’s plate reader

This is a general protocol that determines the range of detection of the plate reader in order to calculate the required number of A375, RPMI-7951, and OUMS-23 cells to yield 90–95% confluency in 5 days for Protocols 3 and 5.

This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUMS-23 cells).

Each cell line has 5 conditions to be performed with six technical replicates per experiment:

A375 cells:

1600 cells/well

1400 cells/well

1200 cells/well

1000 cells/well

800 cells/well

RPMI-7951 cells

3400 cells/well

3200 cells/well

3000 cells/well

2800 cells/well

2600 cells/well

OUMS-23 cells

3400 cells/well

3200 cells/well

3000 cells/well

2800 cells/well

2600 cells/well

Note:

A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Plate 800 – 1600 A375 cells, 2600 – 3400 RPMI-7951 cells, and 2600 – 3400 OUMS-23 cells in 96 well plates with 100 µl of medium. Incubate for 5 days.

Plate media alone (no cells) in columns 1 and 12.

Plate cells in remaining wells (columns 2–11).

Exclude plating in the first and last row to avoid edge effects and evaporation.

5 days later estimate confluency and determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described:

Add 11 µl/well reagent WST-1 (1:10 dilution).

Incubate cells for 20–30 min.

Shake thoroughly for 1 min on a shaker.

Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. (If reference wavelength is to be determined, a filter >600 nm is recommended)

Exclude rows A-H due to edge effects/evaporation, thus making each seeding six technical replicates.

Calculate viability after background subtraction.

Use starting cell numbers that give ~90–95% confluency in 5 days and is in the linear range of the viability assay.

Original report used 1500 A375 cells, 3000 RPMI-7951 cells, and 3000 OUMS-23 cells.

Data to be collected:

Raw data and background subtracted absorbance at 420–480 nm.

n/a

All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. This All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 3: PLX4720 growth inhibitory analysis in cells expressing elevated MAP3K8

This experiment assesses the effect the RAF inhibitor, PLX4720, has on cellular viability, in cells expressing elevated MAP3K8. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiment reported in Figure 3D.

Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is from a single biological replicate, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

See Power Calculations section for details.

Experiment has 3 cohorts:

Cohort 1: A375 cells

Cohort 2: RPMI-7951 cells

Cohort 3: OUMS-23 cells

Each cohort has 9 conditions to be performed with six technical replicates per experiment:

DMSO (vehicle)

100 µM PLX4720

10 µM PLX4720

1 µM PLX4720

0.1 µM PLX4720

0.01 µM PLX4720

0.001 µM PLX4720

0.0001 µM PLX4720

0.00001 µM PLX4720

Note:

A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Plate number of A375, RPMI-7951, and OUMS-23 cells as determined in Protocol 2 in a 96 well plate with 90 µl of medium per well. Incubate for 24 hr.

Plate media alone (no cells) in columns 1 and 12.

Plate cells in remaining wells (columns 2–11).

Exclude plating in the first and last row to avoid edge effects and evaporation.

One plate is needed for each cell line.

Treat cells with 10 µl of 10X serial dilutions of PLX4720 to yield final dilutions of 100 µM to 10-5 µM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2 and 11). Incubate for 96 hr.

Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO (100 mM to 0.01 µM).

Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock (1 mM to 10-4 µM) that is added directly to the 90 µl of cell/medium.

Final DMSO concentration kept to 0.1%.

Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described:

Add 11 µl/well reagent WST-1 (1:10 dilution).

Incubate cells for 20–30 min.

Shake thoroughly for 1 min on a shaker.

Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. (If reference wavelength is to be determined, a filter >600 nm is recommended)

Exclude rows A-H due to edge effects/evaporation, thus making each cohort six technical replicates, except DMSO (vehicle), which has 12.

Calculate viability as a percentage of control (DMSO (vehicle) cells) after background subtraction.

Determine GI50 value by fitting data using a nonlinear regression curve fit with a sigmoid dose-response curve (four-parameter log-logistic function).

Repeat steps 1–3 independently two additional times.

Data to be collected:

Raw data and background subtracted absorbance at 420–480 nm.

GI50 values of each biological replicate.

Graph of average GI50 values for each condition. (Compare to Figure 3D.)

Statistical Analysis of the Replication Data:

One way ANOVA of GI50 values from A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using Fisher’s LSD test.

A375 cells compared to RPMI-7951 cells.

A375 cells compared to OUMS-23 cells.

Meta-analysis of original and replication attempt effect sizes:

The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

The replication will not include the other BRAF (V600E) cell lines reported in the original paper. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. The seeding density of each cell line was determined in Protocol 2. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells expressing elevated MAP3K8

This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I.

Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

See Power Calculations section for details.

Experiment has five conditions:

Vehicle (DMSO) treated RPMI-7951 cells

20 µM MAP3K8 inhibitor treated RPMI-7951 cells

10 µM MAP3K8 inhibitor treated RPMI-7951 cells

5 µM MAP3K8 inhibitor treated RPMI-7951 cells

1 µM MAP3K8 inhibitor treated RPMI-7951 cells

Each condition will be probed with the following antibodies:

pERK1/2 (T202/Y204)

pERK1/2

pMEK1/2 (S217/221)

MEK1/2

Vinculin

Note:

RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Plate 750,000 RPMI-7951 cells in 6-well plates and incubate for 24–36 hr to achieve log phase growth

Wash twice with 1X PBS and incubate overnight in serum-free growth medium.

Treat cells with 20, 10, 5, and 1 µM MAP3K8 inhibitor or DMSO for 1 hr.

Add drug directly to each well.

Make stocks of MAP3K8 inhibitor at 10 mM in DMSO. (500X dilution for 20 µM)

Dilute stock of MAP3K8 to achieve 1000X final concentration of serial dilution stocks in DMSO (10 mM to 1000 µM).

Final DMSO concentration kept to 0.2%.

Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II.

Add ~100–200 µl 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 µg/µl.

Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed (table-top microfuge) at 4˚C.

Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C.

Separate 35–50 μg of protein per lane on a 10% Tris/Glycine gel with protein ladder following replicating lab’s standard protocol.

Samples run per gel:

Protein molecular weight marker

Vehicle (DMSO) treated RPMI-7951 cells

20 µM MAP3K8 inhibitor treated RPMI-7951 cells

10 µM MAP3K8 inhibitor treated RPMI-7951 cells

5 µM MAP3K8 inhibitor treated RPMI-7951 cells

1 µM MAP3K8 inhibitor treated RPMI-7951 cells

Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to immobilon P following replicating lab’s standard protocol.

After transfer, block non-specific binding and immunoblot membrane with the following primary antibodies for 18 hr at 4˚C following manufacturer recommendations:

mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa

mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa

rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa

rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa

rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa

Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions.

Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions.

Analyze bands with image analysis software, normalize to loading controls, and normalize each dose of MAP3K8 inhibitor to Vehicle (DMSO).

pERK1/2 (T202/Y204) normalized to MEK1/2 (total).

pMEK1/2 (S217/221) normalized to ERK1/2 (total).

Repeat steps 1–10 independently seven additional times.

Data to be collected:

Full image western blot films of all immunoblots including ladder. (Compare to Figure 3I)

Raw data of band analysis and normalized bands for each sample.

Statistical Analysis of the Replication Data:

One-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor with the following analysis using the Bonferroni correction:

One-way ANOVA of pERK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor.

One-sample t-test of pERK1/2 levels of 20 µM treated cells compared to 1 (vehicle treated cells).

One-way ANOVA of pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor.

One-sample t-test of pMEK1/2 levels of 20 µM treated cells compared to 1 (vehicle treated cells).

IC50 values of normalized pERK1/2 and pMEK1/2 levels treated with vehicle or MAP3K8 inhibitor.

Meta-analysis of original and replication attempt effect sizes:

The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 5: Viability analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8

This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-1040 or AZD6244, has on cellular viability, in cells expressing MAP3K8. It utilizes A375 cells expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4E.

Generation of A375 cells expressing MEK1, MEK1DD, and MAP3K8 to be performed once.

Experiment (steps 4–6) to be repeated a total of 4 times for a minimum power of 80%. The original data is from a single biological replicate, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

See Power Calculations section for details.

Experiment has 3 cohorts:

Cohort 1: A375 cells expressing MEK1

Cohort 2: A375 cells expressing MEK1DD

Cohort 3: A375 cells expressing MAP3K8

Each cohort has 6 conditions to be done with six technical repeats per experiment:

Untreated [additional control]

DMSO (vehicle)

10 µM PLX4720

1 µM PLX4720

1 µM PLX4720 + 1 µM AZD6244

1 µM PLX4720 + 1 µM CI-1040

Note:

A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.

293T cells maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Grow and prepare endotoxin-free plasmid constructs according to the manufacturer’s protocol for an endotoxin-free Plasmid Maxiprep Kit.

Viral packaging vectors:

∆8.9 (gag,pol)

VSV-G

DNA construct expression vectors:

pLX-Blast-V5-MEK1

pLX-Blast-V5-MEK1DD

pLX-Blast-V5-MAP3K8

Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids:

pLX_CMV-ORF-fwd primer: 5’-CACCAAAATCAACGGGACTT-3’

pLX-ORF-rev primer: 5’-AGGAGGAGAAAATGAAAGCC-3’

Produce MEK1, MEK1DD, and MAP3K8 lentivirus:

Seed 8 x 105 293T cells per 6 cm dish, incubate.

24 hr later, transfect each plate of 293T cells by adding the following:

1 µg pLX-Blast-V5-MEK1, pLX-Blast-V5-MEK1DD, or pLX-Blast-V5-MAP3K8

900 ng ∆8.9 (gag,pol)

100 ng VSV-G

6 µl FuGene transfection reagent

94 µl OptiMem medium (free of FBS and Pen/Strep). Add OptiMem medium to the aliquots of DNA, then add the FuGene to the OptiMem/DNA mix.

Incubate 30 min at RT, then add to cells.

Harvest virus 72 hr post-transfection, aliquot, and freeze at -80˚C for at least 24 hr before using.

Note: You will need to freeze down enough virus for both Protocols 5 and 6.

Titrate lentivirus:

Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions.

Seeding density is such that cells will be near confluent 3 days after removing virus.

Seed two wells per viral concentration for each virus (total wells = 30).

Add polybrene (4–10 µg/ml final concentration) to plates, swirl to mix, then infect cells with varying concentrations of virus (1:5, 1:10, 1:12, 1:15, and 1:20) in duplicate (i.e. two wells per viral concentration).

Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C.

Add virus to the wells and swirl to mix.

Spin 6-well plates at 2250 RPM for 30 min at 37˚C.

Incubate cells overnight with virus, then change medium the following morning.

Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 µg/ml blasticidin.

After 5–7 days of selection, count the cells in all wells and divide the counts with blasticidin by the non-selected control for each viral dilution.

Use the lowest viral dilution that yields an 85–95% ratio of selected/non-selected cells (originally observed to be in the 1:8 – 1:15 range).

Infect A375 cells with viral supernatant:

Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions.

Seed three wells per virus. (total wells = 9).

Add polybrene (4–10 µg/ml final concentration) to plates, swirl to mix, then infect cells with dilution determined by titration protocol (step 4 above).

Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C.

Add virus to the wells and swirl to mix.

Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and polybrene.

Spin 6-well plates at 2250 RPM for 30 min at 37˚C.

Incubate cells overnight with virus, then change medium the following morning.

Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 µg/ml blasticidin.

Two wells without selection and one well with blasticidin for each virus.

After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin-treated well to calculate infection efficiency.

Divide the counts with blasticidin by the non-selected control.

After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 µl. Incubate for 24 hr.

Plate media alone (no cells) in columns 1,2,11, and 12.

Plate cells in remaining wells (columns 3–10).

Exclude plating in the first and last row to avoid edge effects and evaporation.

One plate is needed for each of the A375 infected cells.

Treat cells with 10 µl of 10X dilutions of PLX4720 with or without AZD6244 or CI-1040 to yield appropriate final concentrations (columns 5 through 8), or treat with DMSO (vehicle) control (columns 4 and 9). Add 10 µl medium for untreated control (columns 3 and 10). Incubate for 96 hr.

Dilute stock of PLX4720 at 1000X final concentration in DMSO (10 mM and 1 mM).

Dilute stock of AZD6244 and CI-1040 at 1000X final concentration in DMSO (1 mM).

Dilute 1000X dilution stocks 1:100 in complete growth medium to yield a 10X stock of appropriate treatment.

Final DMSO concentration kept to 0.2%.

Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described:

Add 11 µl/well reagent WST-1 (1:10 dilution).

Incubate cells for 20–30 min.

Shake thoroughly for 1 min on a shaker.

Measure the absorbance against a background control as blank using a microplate reader at 420–480 nm. (If reference wavelength is to be determined, a filter > 600 nm is recommended)

Exclude rows A-H due to edge effects/evaporation, thus making each cohort six technical replicates, except DMSO (vehicle) and untreated control, which has 12.

Calculate viability as a percentage of control (DMSO(vehicle) cells) after background subtraction.

Repeat steps 5–8 independently three additional times.

Data to be collected:

Raw counts and titration percentages (step 2 above).

Raw counts and infection efficiency (step 3 above).

Raw data and background subtracted absorbance at 420–480 nm.

Relative viability of A375 cells expressing MEK1, MEK1DD, and MAP3K8 as a percentage of DMSO treatment for each cell line. (Compare to Figure 4E.)

Sample for additional protocol:

MEK1, MEK1DD, and MAP3K8 lentivirus for further use (Protocol 6).

Statistical Analysis of the Replication Data:

One-way ANOVA of normalized viability of A375 cells expressing MAP3K8 treated with vehicle, 1 µM PLX4720, 1 µM PLX4720 + CI-1040, 1 µM PLX4720 + AZD6244, or 10 µM PLX4720 with the following planned comparisons using the Bonferroni correction:

1 µM PLX4720 treatment compared to 1 µM PLX4720 + CI-1040.

1 µM PLX4720 treatment compared to 1 µM PLX4720 + AZD6244.

10 µM PLX4720 treatment compared to 1 µM PLX4720 + CI-1040.

10 µM PLX4720 treatment compared to 1 µM PLX4720 + AZD6244.

Two-way ANOVA of normalized viability of A375 cells expressing MEK1 or MEK1DD treated with vehicle, 1 µM PLX4720, 1 µM PLX4720 + CI-1040, 1 µM PLX4720 + AZD6244, or 10 µM PLX4720.

Meta-analysis of original and replication attempt effect sizes:

The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. The seeding density of the A375 cell line was determined in Protocol 2. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1 as described in Protocol 6. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 6 MAPK pathway analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8

This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-1040 or AZD6244, has on the MAPK pathway, as analyzed via Western blot. It utilizes A375 cells expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4F.

Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

See Power Calculations section for details.

Experiment has 3 cohorts:

Cohort 1: A375 cells expressing MEK1

Cohort 2: A375 cells expressing MEK1DD

Cohort 3: A375 cells expressing MAP3K8

Each cohort has 4 conditions:

DMSO (vehicle)

1 µM PLX4720

1 µM PLX4720 + 1 µM AZD6244

1 µM PLX4720 + 1 µM CI-1040

Each condition will be probed with the following antibodies:

pERK1/2 (T202/Y204)

ERK1/2

V5 [additional]

Vinculin

MEK1/2 [additional]

MAP3K8 [additional]

Note:

A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2.

Cells will be sent for mycoplasma testing and STR profiling.

Infect A375 cells with viral supernatant:

Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions.

Seed six wells per virus. (total wells = 18).

Add polybrene (4–10 µg/ml final concentration) to plates, swirl to mix, then infect cells following Protocol 5, step 5 using dilution determined by titration protocol (Protocol 5, step 4).

Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 µg/ml blasticidin.

Five wells without selection and one well with blasticidin for each virus.

After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin-treated well to calculate infection efficiency.

Divide the counts with blasticidin by the non-selected control.

72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 µM PLX4720 with or without 1 µM AZD6244, 1 µM CI-1040, or DMSO. Incubate for 24 hr.

Add drugs directly to each well using a 1000X stock (in DMSO).

Final DMSO concentration kept to 0.2%.

Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II.

Add ~100–200 µl 1% NP-40 lysis buffer to ensure that protein concentration is between 2–3 µg/µl.

Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed (table-top microfuge) at 4˚C.

Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature at 88˚C.

Separate 35–50 μg of protein per lane on a 10% Tris/Glycine gel with protein ladder following replicating lab’s standard protocol.

Samples run per gel(s):

Protein molecular weight marker

Vehicle (DMSO) treated A375 cells expressing MEK1

1 µM PLX4720 treated A375 cells expressing MEK1

1 µM PLX4720 and 1 µM AZD6244 treated A375 cells expressing MEK1

1 µM PLX4720 and 1 µM CI-1040 treated A375 cells expressing MEK1

Vehicle (DMSO) treated A375 cells expressing MEK1DD

1 µM PLX4720 treated A375 cells expressing MEK1DD

1 µM PLX4720 and 1 µM AZD6244 treated A375 cells expressing MEK1DD

1 µM PLX4720 and 1 µM CI-1040 treated A375 cells expressing MEK1DD

Vehicle (DMSO) treated A375 cells expressing MAP3K8

1 µM PLX4720 treated A375 cells expressing MAP3K8

1 µM PLX4720 and 1 µM AZD6244 treated A375 cells expressing MAP3K8

1 µM PLX4720 and 1 µM CI-1040 treated A375 cells expressing MAP3K8

Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to immobilon P following replicating lab’s standard protocol.

After transfer, block non-specific binding and immunoblot membrane with the following primary antibodies for 18 hr at 4˚C following manufacturer recommendations:

mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa

mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa

mouse anti-V5-HRP; use at 1:5000 dilution; 45 kDa for MEK1, 52, 58 kDa for MAP3K8

rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa

rabbit anti-MAP3K8; use at 1:500 dilution; 52, 58 kDa

rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa

Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer’s instructions.

Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer’s instructions.

Analyze bands with image analysis software and normalize to loading controls.

pERK1/2 (T202/Y204) normalized to MEK1/2 (total).

V5 (tag on exogenous proteins) normalized to Vinculin.

Repeat steps 1–9 independently two additional times.

Data to be collected:

Raw counts and infection efficiency (step 1 above).

Full image western blot films of all immunoblots including ladder. (Compare to Figures 2A and 4F.)

Raw data of band analysis and normalized bands for each sample.

Statistical Analysis of the Replication Data:

One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8 treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 with the following planned comparisons using the Bonferroni correction:

PLX4720 treatment compared to PLX4720 + CI-1040.

PLX4720 treatment compared to PLX4720 + AZD6244.

Two-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MEK1 or MEK1DD treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244.

Meta-analysis of original and replication attempt effect sizes:

The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1% NP-40. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Power Calculations

For additional details on power calculations, please see analysis scripts and associated files on the Open Science Framework:

https://osf.io/sptzv/

Protocol 1

The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. To identify a suitable sample size, power calculations were performed using different levels of relative variance.

Summary of estimated original data reported in Figure 3E:

F test, ANOVA: Fixed effects, special, main effects and interactions, Bonferorni’s correction: alpha error = 0.00833

Test family

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

Partial η2 calculated from (Lakens, 2013).

Comparisons are between DMSO and all PLX4720 doses (10, 1, and 0.1 µM)

In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 2

Not applicable

Protocol 3

The original data is from a single biological replicate. To identify a suitable sample size, power calculations were performed using different levels of relative variance.

Summary of estimated original data reported in Figure 3D:

2 tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

In order to produce quantitative replication data, we will run the experiment three times. Each time we will calculate the GI50 value. We will determine the standard deviation of GI50 values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 4

The original data does not indicate the error associated with multiple biological replicates. To identify a suitable sample size, power calculations were performed using different levels of relative variance.

Summary of original data reported in Figure 3I:

IC50 values performed with R software, version 3.2.1 (Team, 2015).

F test, ANOVA: Fixed effects, omnibus, one-way, Bonferroni’s correction: alpha error = 0.025

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

ANOVA F test statistic and partial η2 performed with R software, version 3.2.1 (Team, 2015).

Partial η2 calculated from (Lakens, 2013).

t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.025

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

In order to produce quantitative replication data, we will run the experiment eight times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 5

The original data is from a single biological replicate. To identify a suitable sample size, power calculations were performed using different levels of relative variance.

Summary of original data reported in Figure 4E (provided by authors)

F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05.

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

ANOVA F test statistic and partial η2 performed with R software, version 3.2.1 (Team, 2015).

MAP3K8 values

2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha error = 0.0125

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05.

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

ANOVA F test statistic and partial η2 performed with R software, version 3.2.1 (Team, 2015).

MEK1 and MEK1DD values

In order to produce quantitative replication data, we will run the experiment four times. Each time we will quantify relative viability. We will determine the standard deviation of viability values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 6

The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible.

Summary of estimated original data reported in Figure 4F:

F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05.

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

ANOVA F test statistic and partial η2 performed with R software, version 3.2.1 (Team, 2015).

2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha error = 0.025

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05.

Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007).

ANOVA F test statistic and partial η2 performed with R software, version 3.2.1 (Team, 2015).

In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation" for consideration by eLife. Your submission has been evaluated by Tony Hunter who has reviewed the protocols presented. We would ask to make to address the following comments before peer review:

1) In Protocol 4, the authors state "This experiment assesses the effect of the COT kinase inhibitor, TPL2, has on the MAPK pathway, in cells expressing elevated COT, as analyzed via Western blot." This leads me to believe that the authors are unaware that TPL2 is another name for COT, which is an ongoing confusion in the literature, and, from the start of the report, they need to clarify that COT and TPL2 are the same thing, both of which have the formal name MAP3K8. The authors of the paper being replicated obtained the TPL2/COT/MAP3K8 inhibitor from EMD Millipore, whereas the authors plan to obtain the TPL2/COT inhibitor from Santa Cruz rather than EMD Millipore. However, two compounds from the two companies have slightly different structures (i.e. EMD sells: 4-(cycloheptylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine, whereas Santa Cruz sell: 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine, as their TPL2/COT inhibitor (sc-204351). This may not make a difference, but the authors will need to validate sc-204351 to show it has the expected ability to inhibit TPL2/COT kinase activity at the concentrations they are planning to use.

2) In Protocols 5 and 6, the authors plan to generate A375 cells expressing exogenous MEK1, MEK1DD and MAP3K8/COT using lentivirus expression vector infection and blasticidin drug selection. However, they do not plan to ensure that the infected cells are actually expressing the kinase of interest at elevated levels, and this is obviously essential to do, so that the consequences of treatment with PLX4720 can be properly interpreted.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your work entitled "Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation" for peer review at eLife. We apologize for the long delay in reaching this decision, but there was difficulty in finding qualified reviewers. Your submission has been favorably evaluated by Tony Hunter (Senior Editor) and two reviewers.

Reviewer 2 was satisfied, and therefore I have only included below the comments of Reviewer 1. As you will see the reviewer's comments mostly pertain to improving the quality of the experiments and the validity of the conclusions that can be drawn from them. Since the goal of a Registered Report is to replicate the original studies, you are not required to include the additional experiments, but if you believe that any of the suggested experiments would strengthen the conclusions and can readily be incorporated, perhaps as separate data panels, then you could do so. Please let me know your plans for proceeding with the experimental studies for the Registered Report.

Reviewer 1:

The Registered Report aims to repeat key experiments from the 2010 study of Johnanessen et al. which proposed that COT/TPL2 can drive the resistance of B-RAF[V600E] transformed cell lines to a selective RAF kinase inhibitor. COT/TPL2 was identified in an overexpression screen for genes that promote survival of A375 cultured with the specific RAF inhibitor PLX4720. This initial result is unsurprising given that is well established (in papers published over a decade earlier) that COT/TPL2 functions as a MKK1/2 kinase (similar to RAF kinases) and is constitutively active when overexpressed.

Proposed experiments:

The original paper proposed that overproduction of endogenous COT/TPL2 drives resistance of malignant melanomas to PLX4720. However, this conclusion was largely based on experiments with one malignant melanoma line, RPMI-7951 in which COT/TPL2 levels are relatively high. The impact of the study would have been greater if other RAF[V600E] transformed cell lines with COT/TPL2 overexpression had been analyzed in detail as per RPMI-7951 cells.

Protocol 1 (Figures 3B and 3E)

The effect of a range of concentrations of PLX4720 will be tested on the A375 and RPMI-7951 cell lines. Activation of the MKK1/2 > ERK1/2 MAP kinase pathway will be monitored by immunoblotting.

The original Figure 3E tests the effect of PLX4720 on A375, OUMS-23 and RPMI-7951. For the replication study, it is important that OUMS-23 cells, which express very high levels of COT/TPL2, are added to the proposed experiments to determine whether ERK1/2 phosphorylation is maintained in these cells after PLX4720 treatment. As noted above, a limitation of the original study was the dependence on very few cell lines, making generalization of results difficult.

Cell lysate will be resolved by SDS-PAGE and immunoblotted for pERK1/2, total ERK1/2, pMKK1/2, total MKK1/2, COT/TPL-2 and actin. From the protocol, three combinations of antibodies will be tested, presumably in three separate gels (although this is not clear). In our experience, probing blots with pERK1/2 antibody interferes with subsequent probing for total ERK1/2, even after antibody stripping. Consequently, it is better to probe for pERK1/2 and MKK1/2 on one blot and for pMKK1/2 and ERK1/2 on another.

It is planned to use HRP-coupled secondary antibodies and ECL for immunoblotting. However, the linear range of ECL is relatively small and it would be much better to quantify bands using IR Dye-labeled secondary antibodies and a LI-COR scanner. This was also true when the original experiments were carried out.

Protocol 3

This protocol will test the effect of PLX4720 on the viability of A375 and RPMI-7951 cells. However, the experiment shown in Figure 3D of the original paper tested the effect of PLX4720 on a panel of cells lines, with low or high COT/TPL2 expression. Since the authors are making general claims about the role of COT/TPL2 in resistance to RAF inhibitors, OUMS-23 should be analyzed as well. The inclusion of a cell line with low COT/TPL2 levels, similar to A375, would also be preferable.

Protocol 4

The authors have used a Wyeth COT/TPL2 small molecule inhibitor to determine whether COT/TPL2 signaling is required for MKK1/2 and ERK1/2. Our experience some of the early Wyeth compounds is that they have clear off-target effects (revealed by testing their effect on cytokine production in LPS-stimulated Map3k8-/- macrophages). The significance of the experiment shown in Figure 3I therefore is highly questionable. In addition, since the effects of the inhibitor on are only partial, pMKK1/2 and pERK1/2 levels in the original study should have been quantified from multiple experiments and differences tested for statistical significance (as noted in the Registered Report proposal). In view of the limitations of the COT/TPL2 inhibitor experiment, it is important that the siRNA knockdown experiment in Figure 3H is repeated. For this, it is essential to correlate reduction in pERK1/2 levels with the degree of COT/TPL2 knockdown achieved with shCOT-1 and shCOT-2.

See comments on Protocol 2 regarding lysis buffer, immunoblotting etc.

Comment

In Figure 4C, why are A375 cells expressing MEK1DD sensitive to RAF265 inhibitor? Similarly, with SKMEL28 cells expressing MEK1DD. I could find no reference to these results in the text. I would have expected MEK1DD expression in A375 cells to bypass the requirement for constitutive RAF signaling. Am I missing something?

1) In Protocol 4, the authors state "This experiment assesses the effect of the COT kinase inhibitor, TPL2, has on the MAPK pathway, in cells expressing elevated COT, as analyzed via Western blot." This leads me to believe that the authors are unaware that TPL2 is another name for COT, which is an ongoing confusion in the literature, and, from the start of the report, they need to clarify that COT and TPL2 are the same thing, both of which have the formal name MAP3K8. The authors of the paper being replicated obtained the TPL2/COT/MAP3K8 inhibitor from EMD Millipore, whereas the authors plan to obtain the TPL2/COT inhibitor from Santa Cruz rather than EMD Millipore. However, two compounds from the two companies have slightly different structures (i.e. EMD sells: 4-(cycloheptylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine, whereas Santa Cruz sell: 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine, as their TPL2/COT inhibitor (sc-204351). This may not make a difference, but the authors will need to validate sc-204351 to show it has the expected ability to inhibit TPL2/COT kinase activity at the concentrations they are planning to use.

Thank you for the suggestion about the protein name. We have gone through the manuscript and defined in the Introduction all the names used for the protein of interest and for consistency are using the formal name MAP3K8 from that point on. We used the same name that was used in the original publication for each experiment/figure/reagent in an attempt to make the comparison easier to follow, but agree it is better to not add to the ongoing confusion in the literature.

Regarding the inhibitor, we have switched the inhibitor that will be used to the EMD source to minimize differences from the original experiment. However, the EMD source originally used, and now planned to be used in the replication attempt, is the same compound as the Santa Cruz source we had originally suggested (4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine; CAS number: 871307-18-5. EMD also has the type II MAP3K8 kinase inhibitor (4-(cycloheptylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine; CAS number: 1186649-59-4), however this molecule is not being utilized in the replication attempt as it was not used in the original experiment.

2) In Protocols 5 and 6, the authors plan to generate A375 cells expressing exogenous MEK1, MEK1

The expression of the exogenous proteins will be assessed by western blot using an antibody against the V5 tag, which is included in the vectors for expression of MEK1, MEK1DD, and MAP3K8. We have more clearly described this in the revised manuscript. Additionally, in the revised manuscript we are also including antibodies against MAP3K8 and MEK1 to assess the relative degree of elevated protein levels by western blot.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Reviewer 2 was satisfied, and therefore I have only included below the comments of Reviewer 1. As you will see the reviewer's comments mostly pertain to improving the quality of the experiments and the validity of the conclusions that can be drawn from them. Since the goal of a Registered Report is to replicate the original studies, you are not required to include the additional experiments, but if you believe that any of the suggested experiments would strengthen the conclusions and can readily be incorporated, perhaps as separate data panels, then you could do so. Please let me know your plans for proceeding with the experimental studies for the Registered Report. Reviewer 1:

Proposed experiments: The original paper proposed that overproduction of endogenous COT/TPL2 drives resistance of malignant melanomas to PLX4720. However, this conclusion was largely based on experiments with one malignant melanoma line, RPMI-7951 in which COT/TPL2 levels are relatively high. The impact of the study would have been greater if other RAF[V600E] transformed cell lines with COT/TPL2 overexpression had been analyzed in detail as per RPMI-7951 cells. Protocol 1 (Figures 3B and 3E) The effect of a range of concentrations of PLX4720 will be tested on the A375 and RPMI-7951 cell lines. Activation of the MKK1/2 > ERK1/2 MAP kinase pathway will be monitored by immunoblotting.

The original Figure 3E tests the effect of PLX4720 on A375, OUMS-23 and RPMI-7951. For the replication study, it is important that OUMS-23 cells, which express very high levels of COT/TPL2, are added to the proposed experiments to determine whether ERK1/2 phosphorylation is maintained in these cells after PLX4720 treatment. As noted above, a limitation of the original study was the dependence on very few cell lines, making generalization of results difficult.

Thank you for this suggestion. We agree and have added the OUMS-23 cell line in the revised manuscript. We have also adjusted the analysis plans and power calculations to reflect the analysis of OUMS-23 cells similar to A375 and RPMI-7951.

Cell lysate will be resolved by SDS-PAGE and immunoblotted for pERK1/2, total ERK1/2, pMKK1/2, total MKK1/2, COT/TPL-2 and actin. From the protocol, three combinations of antibodies will be tested, presumably in three separate gels (although this is not clear). In our experience, probing blots with pERK1/2 antibody interferes with subsequent probing for total ERK1/2, even after antibody stripping. Consequently, it is better to probe for pERK1/2 and MKK1/2 on one blot and for pMKK1/2 and ERK1/2 on another.

We have clarified the protocols where multiple gels are run to indicate the intention of running three separate gels. We have also changed the probing of the blots to reflect this strategy. As a result, we have also changed the cat # of the total ERK1/2 and MEK1/2 antibodies to a different species (opposite what pERK1/2 and pMEK1/2 are) in addition to the normalization strategy to reflect what is probed on the same blot.

It is planned to use HRP-coupled secondary antibodies and ECL for immunoblotting. However, the linear range of ECL is relatively small and it would be much better to quantify bands using IR Dye-labeled secondary antibodies and a LI-COR scanner. This was also true when the original experiments were carried out.

We agree that IR Dye-labeled secondary antibodies and a LI-COR scanner are ideal and the linear range of ECL is relatively small. While the lab is not equipped with a LI-COR scanner we will make all the exposures from ECL publicly available. There is also the possibility the lab might be able to access a nearby scanner. We have added a statement in the manuscript to indicate that if a LI-COR scanner is available it will be used instead of the ECL approach, with the HRP coupled secondary antibodies switched for the appropriate IR Dye-labeled secondary antibodies. This does not impact the proposed analysis plan. If the antibody detection is performed with the LI-COR scanner, it will also be made transparent in the Replication Study.

Protocol 3 This protocol will test the effect of PLX4720 on the viability of A375 and RPMI-7951 cells. However, the experiment shown in Figure 3D of the original paper tested the effect of PLX4720 on a panel of cells lines, with low or high COT/TPL2 expression. Since the authors are making general claims about the role of COT/TPL2 in resistance to RAF inhibitors, OUMS-23 should be analyzed as well. The inclusion of a cell line with low COT/TPL2 levels, similar to A375, would also be preferable.

Thank you for this suggestion. We agree and have added the OUMS-23 cell line in the revised manuscript for Protocol 2 as well as Protocol 3. We have also adjusted the analysis plans and power calculations to reflect the analysis of OUMS-23 cells similar to A375 and RPMI-7951.

Protocol 4 The authors have used a Wyeth COT/TPL2 small molecule inhibitor to determine whether COT/TPL2 signaling is required for MKK1/2 and ERK1/2. Our experience some of the early Wyeth compounds is that they have clear off-target effects (revealed by testing their effect on cytokine production in LPS-stimulated Map3k8-/- macrophages). The significance of the experiment shown in Figure 3I therefore is highly questionable. In addition, since the effects of the inhibitor on are only partial, pMKK1/2 and pERK1/2 levels in the original study should have been quantified from multiple experiments and differences tested for statistical significance (as noted in the Registered Report proposal). In view of the limitations of the COT/TPL2 inhibitor experiment, it is important that the siRNA knockdown experiment in Figure 3H is repeated. For this, it is essential to correlate reduction in pERK1/2 levels with the degree of COT/TPL2 knockdown achieved with shCOT-1 and shCOT-2.

See comments on Protocol 2 regarding lysis buffer, immunoblotting etc.

We agree that including the shCOT experiment would be of interest considering the limitations of the COT/TPL2 inhibitor experiment and by excluding it limits the scope of what can be interpreted from this replication attempt, but we are attempting to identify a balance of breadth of sampling for general inference with sensible investment of resources on replication projects. While the COT/TPL2 small molecular inhibitor has off-target effects, our anticipation is that the proposed quantification and statistical tests will further evaluate whether this inhibitor has an effect on pMEK1/2 and pERK1/2 levels, whether directly or indirectly through COT/TPL2.

Comment In Figure 4C, why are A375 cells expressing MEK1

We agree with the reviewer that this seems to be an interesting observation made by the original lab even though it wasn’t discussed and should be investigated by future replication efforts.

Reagent	Type	Manufacturer	Catalog #	Comments
RPMI medium with L-glutamine	Cell culture	Sigma	R8758	Replaces Corning cat no. 10-040-CV. Communicated by authors.
MEM with L-glutamine	Cell culture	Sigma	M4655-500ML	Replaces Corning cat. No. 10-010-CV. Communicated by authors.
Fetal bovine serum (FBS)	Cell culture	Life Technologies	12483-020	Replaces Corning brand. Communicated by authors.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand. Communicated by authors.
A375 cells	Cell line	ATCC	CRL-1619	Original brand not specified.
RPMI-7951 cells	Cell line	ATCC	HTB-66
OUMS-23 cells	Cell line	JCRB	JCRB1022	Original brand not specified.
6-well plates	Labware	Greiner bio-one	657 160	Original brand not specified.
Phosphate buffered saline (PBS)	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
PLX4720	Inhibitor	Selleck Chemicals	S1152	Replaces Symansis brand.
DMSO	Chemical	Sigma	D4540	Original brand not specified.
NP-40 buffer	Buffer	Life tech	FNN0021	Original brand not specified.
Protease inhibitors	Inhibitor	Roche	04693116001	Original catalog # not specified.
Phosphatase inhibitor cocktail I	Inhibitor	Sigma	P2850	Replaces CalBioChem brand.
Phosphatase inhibitor cocktail II	Inhibitor	Sigma	P5726	Replaces CalBioChem brand.
Cell scraper	Labware	Sarstedt	83.1830	Original brand not specified.
BCA kit	Reporter assay	Pierce	23227	Original catalog # not specified. Communicated by authors.
Dithiothreitol (DTT)	Chemical	Biobasic	DB0058	Original brand not specified.
Sample buffer	Buffer	Abm	G031	Replaces Invitrogen brand.
Protein molecular weight ladder	Western materials	Abm	G252, G494	Original brand not specified.
10% Tris/Glycine gel; 10 well, 1.0 mm thick	Western materials	Abm	Internal	Replaces Invitrogen brand.
Running buffer	Buffer	Abm	Internal	Original brand not specified.
Immobilon P	Western materials	Thermofisher	IPVH00010	Original brand not specified.
Transfer buffer	Buffer	Abm	Internal	Original brand not specified.
Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody (clone E10)	Antibodies	Cell Signaling	9106	Use at 1:1000 dilution. Original catalog # not specified.
Rabbit anti-pMEK1/2 (S217/221) (clone 41G9) antibody	Antibodies	Cell Signaling	9154	Use at 1:11000 dilution. Original catalog # not specified.
Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody	Antibodies	Cell Signaling	4696	Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors.
Rabbit anti-MEK1/2 (clone D1A5) antibody	Antibodies	Cell Signaling	8727	Use at 1:1000 dilution. Original catalog # not specified.
Rabbit anti-MAP3K8 (clone M-20) antibody	Antibodies	Santa Cruz	sc-720	Use at 1:500 dilution. Communicated by authors.
Mouse anti-ß-Actin (clone C4) antibody	Antibodies	Santa Cruz	sc-47778	Use at 1:100 – 1:1000 dilution. Original catalog # not specified.
Anti-rabbit IgG – HRP conjugated antibody	Antibodies	Cell Signaling	7074	Use at 1:1000 dilution. Original catalog # not specified.
Anti-mouse IgG – HRP conjugated antibody	Antibodies	Cell Signaling	7076	Use at 1:1000 dilution. Original catalog # not specified.
Chemiluminescent reagent	Western materials	Life Technologies	WP20005	Replaces Pierce brand.

Protocol 1 Western Blot Antibody
	POI		Loading Control
Independent Gels	Description	Working Conc.	Description	Working Conc.
1	Mouse anti-pERK1/2 (T202/Y204) (42, 44kDa)	1:1000	Rabbit anti-MEK1/2 (45 kDa)	1:1000
2	Rabbit anti-pMEK1/2 (S217/221) (45 kDa)	1:1000	Mouse anti-ERK1/2 (42, 44 kDa)	1:1000
3	Rabbit anti-MAP3K8 (52, 58 kDa)	1:500	Mouse anti-ß-Actin (43 kDa)	1:100 – 1:1000

Reagent	Type	Manufacturer	Catalog #	Comments
RPMI medium with L-glutamine	Cell culture	Sigma	R8758	Replaces Corning cat no. 10-040-CV.
MEM with L-glutamine	Cell culture	Sigma	M4655-500ML	Replaces Corning cat. no. 10-010-CV.
FBS	Cell culture	Life Technologies	12483-020	Replaces Corning brand.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand.
A375 cells	Cell line	ATCC	CRL-1619	Original brand not specified.
RPMI-7951 cells	Cell line	ATCC	HTB-66
OUMS-23 cells	Cell line	JCRB	JCRB1022	Original brand not specified.
PBS	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
96 well clear plates	Labware	Sarstedt	83.3924	Original brand not specified.
WST1 viability assay	Reporter assay	Roche	11644807001	Original catalog # not specified.
Microplate reader (420–480 nm)	Instrument	Molecular Devices	abm	Original brand not specified.

Reagent	Type	Manufacturer	Catalog #	Comments
RPMI medium with L-glutamine	Cell culture	Sigma	R8758	Replaces Corning cat no. 10-040-CV.
MEM with L-glutamine	Cell culture	Sigma	M4655-500ML	Replaces Corning cat. no. 10-010-CV.
FBS	Cell culture	Life Technologies	12483-020	Replaces Corning brand.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand.
A375 cells	Cell line	ATCC	CRL-1619	Original brand not specified.
RPMI-7951 cells	Cell line	ATCC	HTB-66
OUMS-23 cells	Cell line	JCRB	JCRB1022	Original brand not specified.
PBS	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
96 well clear plates	Labware	Sarstedt	83.3924	Original brand not specified.
PLX4720	Inhibitor	Selleck Chemicals	S1152	Replaces Symansis brand.
DMSO	Chemical	Sigma	D4540	Original brand not specified.
WST1 viability assay	Reporter assay	Roche	11644807001	Original catalog # not specified.
Microplate reader (420-480 nm)	Instrument	Molecular Devices	abm	Original brand not specified.

Reagent	Type	Manufacturer	Catalog #	Comments
MEM with L-glutamine	Cell culture	Sigma	M4655-500ML	Replaces Corningcat. no. 10-010-CV
FBS	Cell culture	Life Technologies	12483-020	Replaces Corning brand.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand.
RPMI-7951 cells	Cell line	ATCC	HTB-66
PBS	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
6 well plates	Labware	Greiner bio-one	657 160	Original brand not specified.
MAP3K8 kinase inhibitor	Inhibitor	EMD	616373
DMSO	Chemical	Sigma	D4540	Original brand not specified.
NP-40 buffer	Buffer	Life Technologies	FNN0021	Original brand not specified.
Protease inhibitors	Inhibitor	Roche	04693116001	Original catalog # not specified.
Phosphatase inhibitor cocktail I	Inhibitor	Sigma	P2850	Replaces CalBioChem brand.
Phosphatase inhibitor cocktail II	Inhibitor	Sigma	P5726	Replaces CalBioChem brand.
Cell scraper	Labware	Sasrstedt	83.1830	Original brand not specified.
BCA kit	Reporter assay	Pierce	23227	Original catalog # not specified. Communicated by authors.
DTT	Chemical	Biobasic	DB0058	Original brand not specified.
Sample buffer	Buffer	abm	G031	Replaces Invitrogen brand.
Protein molecular weight ladder	Western materials	abm	G252, G494	Original brand not specified.
10% Tris/Glycine gel; 10 well, 1.0 mm thick	Western materials	abm	internal	Replaces Invitrogen brand.
Running buffer	Buffer	abm	internal	Original brand not specified.
Immobilon P	Western materials	Thermofisher	IPVH00010	Original brand not specified.
Transfer buffer	Buffer	abm	internal	Original brand not specified.
Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody	Antibodies	Cell Signaling	9106	Use at 1:1000 dilution. Original catalog # not specified.
Rabbit anti-pMEK1/2 (S217/221) (clone 41G9) antibody	Antibodies	Cell Signaling	9154	Use at 1:11000 dilution. Original catalog # not specified.
Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody	Antibodies	Cell Signaling	4696	Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors.
Rabbit anti-MEK1/2 (clone D1A5) antibody	Antibodies	Cell Signaling	8727	Use at 1:1000 dilution. Original catalog # not specified.
Rabbit anti-Vinculin antibody	Antibodies	Sigma	V4139	Use at 1:20,000 dilution. Original catalog # not specified.
Anti-rabbit IgG – HRP conjugated	Antibodies	Cell Signaling	7074	Use at 1:1000 dilution. Original catalog # not specified.
Anti-mouse IgG – HRP conjugated	Antibodies	Cell Signaling	7076	Use at 1:1000 dilution. Original catalog # not specified.
Chemiluminescent reagent	Western materials	Life Technologies	WP20005	Replaces Pierce brand.

Protocol 4 Western blot antibody combinations
	POI		Loading Control
Independent Gels	Description	Working Conc.	Description	Working Conc.
1	Mouse anti-pERK1/2 (T202/Y204) (42, 44 kDa)	1:1000	Rabbit anti-MEK1/2 (45 kDa)	1:1000
2	Rabbit anti-pMEK1/2 (S217/221) (45 kDa)	1:1000	Mouse anti-ERK1/2 (42, 44 kDa)	1:1000
3			Rabbit anti-Vinculin (116 kDa)	1:20000

Reagent	Type	Manufacturer	Catalog #	Comments
RPMI medium with L-glutamine	Cell culture	Sigma	R8758	Replaces Corning cat no. 10-040-CV.
DMEM medium	Cell culture	Corning	10-013	Original brand not specified.
FBS	Cell culture	Life Technologies	12483-020	Replaces Corning brand.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand.
A375 cells	Cell line	ATCC	CRL-1619	Original brand not specified.
293T cells	Cell line	ATCC	CRL-11268
PBS	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
6 cm plates	Labware	Biolite	11825275	Original brand not specified.
Nucleobond Maxiprep Kit	Kit	Macherey-Nagel	740414	Not originallyspecified
pLX-Blast-V5-MEK1	DNA construct	Provided from original authors
pLX-Blast-V5-MEK1DD	DNA construct	Provided from original authors
pLX-Blast-V5-MAP3K8	DNA construct	Provided from original authors
∆8.9 (gag,pol)	DNA construct	Provided from original authors
VSV-G	DNA construct	Provided from original authors
FuGene HD transfection reagent	Transfection reagent	Promega	E2311	Replaces FuGene6 Roche brand.
OptiMEM medium	Buffer	Life Tech	51985034	Original brand not specified.
6-well plates	Labware	Greiner bio-one	657 160	Original brand not specified.
Polybrene	Cell culture	Sigma	H9268	Original brand not specified.
Blasticidin	Cell culture	Invivogen	ant-bl-1	Original brand not specified.
96 well clear plates	Labware	Sarstedt	83.3924	Original brand not specified.
PLX4720	Inhibitor	Selleck Chemicals	S1152	Replaces Symansis brand.
AZD6244	Inhibitor	Selleck Chemicals	S1008	Original catalog # not specified.
CI-1040	Inhibitor	Selleck Chemicals	S1020	Replaces Shanghai Lechen International Trading Co. brand.
DMSO	Chemical	Sigma	D4540	Original brand not specified.
WST1 viability assay	Reporter assay	Roche	11644807001	Original catalog # not specified.
Microplate reader (420–480 nm)	Instrument	Molecular Devices	abm	Original brand not specified.

Reagent	Type	Manufacturer	Catalog #	Comments
RPMI medium with L-glutamine	Cell culture	Sigma	R8758	Replaces Corning cat no. 10-040-CV.
FBS	Cell culture	Life Technologies	12483-020	Replaces Corning brand.
Pen/strep/glutamine	Cell culture	Abm	G255	Replaces Corning brand.
A375 cells	Cell line	ATCC	CRL-1619	Original brand not specified.
PBS	Buffer	Sigma	D8537-500ML	Original brand not specified.
Trypsin	Cell culture	Sigma	T4049	Original brand not specified.
10 cm plates	Labware	CellStar	664 160	Original brand not specified.
6 well plates	Labware	Greiner bio-one	657 160	Original brand not specified.
Polybrene	Cell culture	Sigma	H9268	Original brand not specified.
Blasticidin	Cell culture	Invivogen	ant-bl-1	Original brand not specified.
PLX4720	Inhibitor	Selleck Chemicals	S1152	Replaces Symansis brand.
AZD6244	Inhibitor	Selleck Chemicals	S1008	Original catalog # not specified.
CI-1040	Inhibitor	Selleck Chemicals	S1020	Replaces Shanghai Lechen International Trading Co. brand.
DMSO	Chemical	Sigma	D4540	Original brand not specified.
NP-40 buffer	Buffer	Life technologies	FNN0021	Original brand not specified.
Protease inhibitors	Inhibitor	Roche	04693116001	Original catalog # not specified.
Phosphatase inhibitor cocktail I	Inhibitor	Sigma	P2850	Replaces CalBioChem brand.
Phosphatase inhibitor cocktail II	Inhibitor	Sigma	P5726	Replaces CalBioChem brand.
Cell scraper	Labware	Sasrstedt	83.1830	Original brand not specified.
BCA kit	Reporter assay	Pierce	23227	Original catalog # not specified. Communicated by authors.
DTT	Chemical	Biobasic	DB0058	Original brand not specified.
Sample buffer	Buffer	abm	G031	Replaces Invitrogen brand.
Protein molecular weight ladder	Western materials	abm	G252, G494	Original brand not specified.
10% Tris/Glycine gel; 10 well, 1.0 mm thick	Western materials	abm	internal	Replaces Invitrogen brand.
Running buffer	Buffer	abm	internal	Original brand not specified.
Immobilon P	Western materials	Thermofisher	IPVH00010	Original brand not specified.
Transfer buffer	Buffer	abm	internal	Original brand not specified.
Mouse anti-pERK1/2 (T202/Y204) (clone E10) antibody	Antibodies	Cell Signaling	9106	Use at 1:1000 dilution. Original catalog # not specified.
Mouse anti-p44/42 MAPK (ERK1/2) (clone L34F12) antibody	Antibodies	Cell Signaling	4696	Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors.
Mouse anti-V5-HRP conjugated antibody	Antibodies	Invitrogen	R961-25	Use at 1:5000 dilution. Original catalog # not specified.
Rabbit anti-Vinculin antibody	Antibodies	Sigma	V4139	Use at 1:20,000 dilution. Original catalog # not specified.
Rabbit anti-MEK1/2 (clone D1A5) antibody	Antibodies	Cell Signaling	8727	Use at 1:1000 dilution. Original catalog # not specified.
Rabbit anti-MAP3K8 (clone M-20) antibody	Antibodies	Santa Cruz	sc-720	Use at 1:500 dilution. Communicated by authors.
Anti-rabbit IgG – HRP conjugated antibody	Antibodies	Cell Signaling	7074	Use at 1:1000 dilution. Original catalog # not specified.
Anti-mouse IgG – HRP conjugated antibody	Antibodies	Cell Signaling	7076	Use at 1:1000 dilution. Original catalog # not specified.
Chemiluminescent reagent	Western materials	Life Technologies	WP20005	Replaces Pierce brand.

Protocol 6 Western blot antibody combinations
	POI		Loading control
Independent Gels	Description	Working Conc.	Description	Working Conc.
1	Mouse anti-pERK1/2 (T202/Y204) (42, 44 kDa)	1:1000	Rabbit anti-MEK1/2 (45 kDa)	1:1000
2	Mouse anti-V5-HRP (45, 52, 58 kDa)	1:5000	Rabbit anti-Vinculin (116 kDa)	1:20000
3	Rabbit anti-MAP3K8 (52, 58 kDa)	1:500	Mouse anti-ERK1/2 (42, 44 kDa)	1:1000

Cell line	PLX4720 concentration (µM)	Normalized pMEK/ERK	Normalized pERK/MEK
A375	0 (DMSO)	1.000	1.000
	10	0.00212	0.0147
	1	0.00283	0.0254
	0.1	0.0981	0.00283
RPMI-7951	0 (DMSO)	1.000	1.000
	10	0.827	1.849
	1	0.380	1.460
	0.1	0.471	1.022
OUMS-23	0 (DMSO)	1.000	1.000
	10	0.0579	0.926
	1	0.197	1.042
	0.1	1.100	1.068

Antibody	Cell line	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
pMEK	A375	F(1,8) = 20781	0.99962	50.9670	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 2128.4	0.99626	16.31121	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 3001.0	0.99734	19.36831	80.0%	12 (4 groups)
pERK	A375	F(1,8) = 21841	0.99963	52.2504	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 582.85	0.98646	8.535621	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 0.8080	0.09173	0.317801	80.0%	12 (4 groups)
15% variance
pMEK	A375	F(1,8) = 369.44	0.97880	6.79560	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 37.839	0.82547	2.174821	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 53.352	0.86960	2.582441	80.0%	12 (4 groups)
pERK	A375	F(1,8) = 388.28	0.97981	6.96672	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 10.362	0.56431	1.138081	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 0.01436	0.001792	0.042371	80.0%	12 (4 groups)
28% variance
pMEK	A375	F(1,8) = 106.68	0.92984	3.64050	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 10.859	0.57581	1.165091	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 15.312	0.65682	1.383451	80.0%	12 (4 groups)
pERK	A375	F(1,8) = 111.43	0.93302	3.73217	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 2.9737	0.27099	0.609691	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 0.04122	0.000515	0.022701	80.0%	12 (4 groups)
40% variance
pMEK	A375	F(1,8) = 51.952	0.86656	2.54835	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 5.3211	0.39945	0.815561	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 7.5026	0.48396	0.968421	80.0%	12 (4 groups)
pERK	A375	F(1,8) = 54.602	0.87221	2.61252	99.9%	12 (4 groups)
	RPMI-7951	F(1,8) = 1.4571	0.15408	0.426781	80.0%	12 (4 groups)
	OUMS-23	F(1,8) = 0.00202	0.000252	0.015891	80.0%	12 (4 groups)

1 This is the calculated effect size using the originally reported value with the indicated variance. Unlike the power calculations to determine sample size, the aim of these sensitivity calculations are not to detect the original effect size, but to understand what effect size could be detected with 80% power and the indicated total sample size of 12. The detectable effect size is 1.29189.

Cell line	GI50 (µM)
A375	0.2276
RPMI-7951	10.862
OUMS-23	8.731

Group 1	Group 2	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
2% variance
A375	RPMI-7951	69.2139	99.9%	2	2
A375	OUMS-23	55.3442	99.9%	2	2
15% variance
A375	RPMI-7951	68.3898	99.9%	2	2
A375	OUMS-23	7.37923	93.3%	2	2
28% variance
A375	RPMI-7951	4.94385	99.2%	3	3
A375	OUMS-23	3.95316	94.4%	3	3
40% variance
A375	RPMI-7951	3.46070	88.0%	3	3
A375	OUMS-23	2.76721	90.0%	4	4

Cell line	MAP3K8 inhibitor concentration (µM)	Normalized pMEK/ERK	Normalized pERK/MEK
RPMI-7951	0 (DMSO)	1.00	1.00
	1	1.35	1.00
	5	1.15	0.92
	10	0.70	0.75
	20	0.36	0.71

IC50 value of normalized pMEK values	IC50 value of normalized pERK values
9.4725	6.4091

Groups	Antibody	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
MAP3K8 inhibitor (1, 5, 10, 20 µM)	pMEK	F(3,8) = 1583.7	0.99832	24.3697	99.9%	8 (4 groups)
	pERK	F(3,8) = 195.33	0.98653	8.55863	99.9%	8 (4 groups)
15% variance
MAP3K8 inhibitor (1, 5, 10, 20 µM)	pMEK	F(3,8) = 28.155	0.91348	3.24931	96.8%	8 (4 groups)
	pERK	F(3,8) = 3.4726	0.56563	1.14114	83.6%	16 (4 groups)
28% variance
MAP3K8 inhibitor (1, 5, 10, 20 µM)	pMEK	F(3,8) = 8.0801	0.75186	1.74070	94.7%	12 (4 groups)
	pERK	F(3,8) = 0.9966	0.27205	0.61133	80.8%	40 (4 groups)
40% variance
MAP3K8 inhibitor (1, 5, 10, 20 µM)	pMEK	F(3,8) = 3.9593	0.59754	1.21849	88.7%	16 (4 groups)
	pERK	F(3,8) = 0.4883	0.15478	0.42793	80.5%	76 (4 groups)

MAP3K8 inhibitor dose	Constant (vehicle)	Antibody	Effect size d	A priori power	Sample size per group
2% variance
20 µM	0 µM	pMEK	88.8889	99.9%	3
		pERK	20.4225	99.9%	3
15% variance
20 µM	0 µM	pMEK	11.8519	99.9%	3
		pERK	2.72300	80.2%	4
28% variance
20 µM	0 µM	pMEK	6.34921	95.1%	3
		pERK	1.45875	86.6%	8
40% variance
20 µM	0 µM	pMEK	4.44444	99.2%	4
		pERK	1.02113	81.1%	12

Cell line	Drug(s)	Relative viability	Technical replicate stdev
MEK1	DMSO	100	5.09
	PLX4720 (10 µM)	5.78	0.67
	PLX4720 (1 µM)	11.32	0.97
	PLX4720 (1 µM) + CI-1040 (1 µM)	10.13	0.60
	PLX4720 (1 µM) + AZD6244 (1 µM)	10.92	0.91
MEK1DD	DMSO	96.96	9.40
	PLX4720 (10 µM)	97.56	6.25
	PLX4720 (1 µM)	92.10	8.23
	PLX4720 (1 µM) + CI-1040 (1 µM)	102.24	8.55
	PLX4720 (1 µM) + AZD6244 (1 µM)	42.44	2.74
MAP3K8	DMSO	100	10.66
	PLX4720 (10 µM)	43.44	3.48
	PLX4720 (1 µM)	62.81	5.05
	PLX4720 (1 µM) + CI-1040 (1 µM)	13.42	2.42
	PLX4720 (1 µM) + AZD6244 (1 µM)	12.52	1.65

Groups	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
All treatments	F(4,25) = 6246.6	0.99900	31.60696	99.9%	10 (5 groups)
15% variance
All treatments	F(4,25) = 111.04	0.94672	4.21509	99.9%	15 (5 groups)
28% variance
All treatments	F(4,25) = 31.868	0.83604	2.25807	99.9%	20 (5 groups)
40% variance
All treatments	F(4,25) = 15.615	0.71416	1.58066	99.9%	25 (5 groups)

Group 1	Group 2	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
2% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	67.45484	99.9%	2	2
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	55.51810	99.9%	2	2
10 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	46.68351	99.9%	2	2
10 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	48.35265	99.9%	2	2
15% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	7.24974	99.4%	3	3
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	7.40241	99.6%	3	3
10 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	6.22447	97.2%	3	3
10 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	6.44702	97.9%	3	3
28% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	3.88379	93.0%	4	4
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	3.96558	94.0%	4	4
10 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	3.33454	82.9%	4	4
10 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	3.45376	85.7%	4	4
40% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	2.71865	82.6%	5	5
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	2.77591	84.3%	5	5
10 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	2.33418	81.5%	6	6
10 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	2.41763	84.5%	6	6

Groups	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
All treatments	F(4,50) = 2716.2	0.99542	14.7409	99.9%	20 (10 groups)
15% variance
All treatments	F(4,50) = 48.287	0.79437	1.96545	99.9%	30 (10 groups)
28% variance
All treatments	F(4,50) = 13.858	0.52576	1.05292	99.9%	40 (10 groups)
40% variance
All treatments	F(4,50) = 6.7904	0.35201	0.73704	98.7%	50 (10 groups)

Cell line	Drug(s)	Normalized pERK/ERK
MEK1	DMSO	1.0000
	PLX4720 (1 µM)	0.0799
	PLX4720 (1 µM) + CI-1040 (1 µM)	0.0021
	PLX4720 (1 µM) + AZD6244 (1 µM)	0.0002
MEK1DD	DMSO	1.0000
	PLX4720 (1 µM)	1.0203
	PLX4720 (1 µM) + CI-1040 (1 µM)	0.3015
	PLX4720 (1 µM) + AZD6244 (1 µM)	0.0166
MAP3K8	DMSO	1.0000
	PLX4720 (1 µM)	0.7689
	PLX4720 (1 µM) + CI-1040 (1 µM)	0.0045
	PLX4720 (1 µM) + AZD6244 (1 µM)	0.0063

Groups	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
All treatments	F(3,8) = 5024.1	0.99947	43.4257	99.9%	8 (4 groups)
15% variance
All treatments	F(3,8) = 89.317	0.97101	5.78735	99.9%	8 (4 groups)
28% variance
All treatments	F(3,8) = 25.633	0.90577	3.10038	99.9%	12 (4 groups)
40% variance
All treatments	F(3,8) = 12.560	0.82487	2.17026	99.9%	16 (4 groups)

Group 1	Group 2	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
2% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	68.8057	99.9%	2	2
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	70.5269	99.9%	2	2
15% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	9.17409	87.8%	2	2
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	9.40358	89.0%	2	2
28% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	4.91469	95.5%	3	3
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	5.03763	96.3%	3	3
40% variance
1 µM PLX4720	1 µM PLX4720 + 1 µM CI1040	3.44028	93.7%	4	4
1 µM PLX4720	1 µM PLX4720 + 1 µM AZD6244	3.52634	94.7%	4	4

Groups	F test statistic	Partial η2	Effect size f	A priori power	Total sample size
2% variance
All treatments	F(3,16) = 1847.2	0.99712	18.6103	99.9%	16 (8 groups)
15% variance
All treatments	F(3,16) = 32.840	0.86029	2.48143	99.9%	16 (8 groups)
28% variance
All treatments	F(3,16) = 9.4247	0.63862	1.32934	99.9%	24 (8 groups)
40% variance
All treatments	F(3,16) = 4.6181	0.46406	0.93053	99.0%	32 (8 groups)