Jeremi Laski1,2, Bipradeb Singha1,2, Xu Wang1,3, Yudith Ramos Valdés1, Olga Collins1, Trevor G Shepherd4,5,6,7,8. 1. The Mary & John Knight Translational Ovarian Cancer Research Unit, Lawson Health Research Institute, London, ON, Canada. 2. Departments of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. 3. West China School of Medicine, Chengdu, Sichuan, China. 4. The Mary & John Knight Translational Ovarian Cancer Research Unit, Lawson Health Research Institute, London, ON, Canada. tshephe6@uwo.ca. 5. Departments of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. tshephe6@uwo.ca. 6. Departments of Oncology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. tshephe6@uwo.ca. 7. Departments of Obstetrics & Gynaecology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. tshephe6@uwo.ca. 8. London Regional Cancer Program, 790 Commissioners Rd. E., Room A4-836, London, ON, N6A 4L6, Canada. tshephe6@uwo.ca.
Abstract
BACKGROUND: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. RESULTS: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. CONCLUSIONS: Our results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.
BACKGROUND: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. RESULTS: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. CONCLUSIONS: Our results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.
Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in women in the developed world, and is responsible for over 70% of all diagnosed cases [1]. The high mortality rates from EOC is most commonly attributed to late-stage diagnosis since its symptoms are shared with those of generalized post-menopausal conditions. In addition, current diagnostic tests are limited to physical pelvic exams, trans-vaginal ultrasound and CA-125 serum tests, all of which have low sensitivity for detection of early disease [2]. Since most EOC cases present with late-stage disease consisting of extensive tumour burden and ascites, treatment requires aggressive surgical debulking procedures coupled with cytotoxic chemotherapy to reduce to minimal residual disease and delay disease progression. Nevertheless, rates of recurrence remain exceptionally high, with relapsed EOC often acquiring chemo-resistance [1]. As such, gaining further understanding of the mechanisms governing late-stage EOC progression and recurrence of chemo-resistant disease is of utmost importance in developing more effective therapeutics [3, 4].During metastasis, EOC cells detach from the primary tumour site, disseminate within the peritoneal fluid of the abdominal cavity, then subsequently re-attach to new sites thereby forming secondary lesions. A unique hallmark of EOC metastasis lies in the process of multicellular spheroid formation thereby affording metastatic cells with enhanced survival and chemo-resistance [5], as well as increased capacity to re-attach and invade the peritoneum [6]. Previous work by our group demonstrated that EOC cells enter a quiescent state within spheroids [7], and they possess reduced metabolic activity with increased AMP-activated protein kinase (AMPK) signaling [8]. AMPK is a conserved serine/threonine heterotrimeric kinase complex acting as a bio-energetic stress sensor in nearly all mammalian systems, primarily to promote cell survival during starvation-like conditions [9]. Following nutrient deprivation, increased levels of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) bind to the gamma subunit of the complex, cause an allosteric shift, and thereby facilitate AMPK phosphorylation at the threonine 172 residue (T172) on the catalytic alpha subunit. AMPK activity often acts as a major bioenergetic regulator to induce catabolic processes while concurrently down-regulating anabolic processes, however, its exact functions are often tissue specific [9].In separate studies, we demonstrated that EOC spheroids upregulate macroautophagy (described here as autophagy) [10, 11], a lysosomal process allowing for the degradation and recycling of intracellular nutrients and damaged organelles [12]. Dual roles have been suggested for autophagy in mediating cancer progression: autophagy can serve tumour suppressive functions particularly during disease initiation, yet a large proportion of studies have demonstrated essential tumour-promoting effects of autophagy in late-stage disease [13]. Tumour cells undergoing cellular stress due to hypoxia, lack of nutrient availability, and during metastasis, hijack native autophagy functions to recycle their intracellular constituents ultimately providing temporary alternative sources of energy and nutrients.As such, AMPK signaling can act as a key link between metabolism homeostasis and autophagy regulation. Although AMPK has several means by which to activate autophagy [14], its potential regulation of autophagy in EOC cells and spheroids has not been determined yet. Herein, we sought to examine whether AMPK signaling mediates autophagy induction in a spheroid model of EOC metastasis. Our results demonstrate that intact AMPK activity is required but not sufficient to promote autophagic flux in EOC spheroids. Treatment of EOC spheroids with the CAMKKβ inhibitor STO-609 potently blocks AMPK activity and autophagic flux leading to reduced cell viability.
Materials and methods
Cell culture
Work was conducted with several established ovarian cancer cell lines: CaOV3, OVCAR3, OVCAR4, OVCAR5, and OVCAR8 (ATCC), COV318 and COV362 (gift from Zia Khan, Western University), all of which are classified as high-grade serous [15, 16], and HeyA8 cells (ATCC). The iOvCa147-MA line was generated by subcutaneous injection of high-grade serous iOvCa147 cells [17] into immune-compromised female mice, isolation and dissociation of the resultant tumour, followed by intraperitoneal injection into subsequent female mice. Malignant ascites fluid was collected aseptically and returned to tissue culture to generate the iOvCa147-MA line. STR analysis was performed by the TCAG Facility (Hospital for Sick Children, Toronto ON) to confirm its identity with the original iOvCa147 cell line. The immortalized human fallopian tube secretory epithelial cell line FT190 [18] (gift from Ronny Drapkin, University of Pennsylvania) was used as a non-malignant cell line control. Cells were cultured in either DMEM/F12 (Invitrogen) for iOvCa147-MA, CaOV3, COV318, COV362, and FT190, or RPMI (Wisent) for OVCAR3, OVCAR4, OVCAR5, OVCAR8, and HeyA8, and supplemented with 10% fetal bovine serum (FBS) (Wisent). Cells were either grown under adherent conditions using tissue culture-treated plastic (Sarstedt) or in suspension using Ultra-Low Attachment (ULA) dishes (Corning) as performed previously [7].
siRNA knockdown
RNA interference-mediated knockdown was achieved using Dharmacon siGenome SMARTpool reagents: Non-targeting control pool #2 (siNT, D-001206-14-05), PRKAA1 (D-001206-14-05) PRKAA2 (M-005361-02-0005). Cells were seeded into 6-well adherent plates at 300,000 cells/well for iOvCa147-MA, or 100,000 cells/well for OVCAR8; the following day siRNA (siNT, or equimolar PRKAA1/2) was transfected according to manufacturer’s instructions as performed previously [8]. Cells were incubated with transfection mixtures for 3 days after which the cells were trypsinized and seeded into ULA dishes for protein isolation and fluorescence microscopy at 48 h.
Protein isolation
Adherent cells were washed with ice-cold phosphate-buffered saline (PBS) and placed in modified radioimmunoprecipitation assay (RIPA) lysis buffer as described previously [11]. Cells were subsequently scraped and left to lyse on ice for 30 min. After high-speed centrifugation, supernatant was collected for protein quantification by Bradford assay (BioRad) and stored at − 80 °C for subsequent use. For protein lysates from spheroids, cell suspensions were collected from ULA plates and centrifuged at 2400 rpm for 3 minutes. The cell pellet was washed with ice-cold PBS and placed in modified RIPA lysis buffer for 30 min on ice and continued as described above.
Immunoblotting
Protein lysates were prepared at 30 μg per sample and resolved by 8 or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% w/v BSA in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20). Membranes were subsequently incubated with protein-specific primary antibodies at a 1:1000 dilution in 5% BSA/TBST and incubated overnight at 4 °C. Following primary antibody incubation, membranes were incubated for 1 h at room temperature with a peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (1:10000 in 5% BSA/TBST). Protein detection was achieved through enhanced chemiluminescence using Luminata Forte (Millipore) and imaging was performed using the Chemidoc™ MP 7 System (BioRad). Densitometric analyses were subsequently performed using ImageLab™ software with tubulin used as a loading control.
Antibodies and other reagents
Primary antibodies were used to detect threonine-172 phosphorylated AMPKα (40H9), total AMPKα (D63G4), p62 (2775S), LC3B (5114S) (Cell Signaling Technology, Danvers, MA), or tubulin (T9026; Sigma, Mississauga, ON). Secondary antibodies used were anti-rabbit horseradish peroxidase (HRP; NA934V Chicago, GE Healthcare) and anti-mouse HRP (NA931V Chicago, GE Healthcare). The following pharmacologic agents were used: Compound C (P5499) and Metformin (D150959-5G) from Sigma (Mississauga, ON), and STO-609 (15325) and Oligomycin (11342) from Cayman Chemical (Ann Arbor, MI) at concentrations indicated in the text.
Generation of mCherry-eGFP-LC3B clones
OVCAR8 cells were plated into 6-well adherent plates at a density of 100,000 cells/well. Cells were transfected with the pBABE-puro-mCherry-eGFP-LC3B plasmid (gift from Jayanta Debnath, Addgene plasmid #22418), using Lipofectamine 2000 reagent according to the manufacturer’s protocol (MAN0009872, Invitrogen, Carlsbad Ca). Following 72 h, cells were placed in puromycin-supplemented media (1 μg/ml) for 72 h. Puromycin-resistant fluorescent clones were selected using cloning rings and expanded clonal populations expressing detectable fluorescence were chosen for subsequent experiments. The COV318 and FT190 cell lines were generated in an identical fashion as described above, however, following puromycin treatment, transfected cells were pooled in suspension conditions using ULA dishes and selected on the basis of high red fluorescence.
Live-cell fluorescence microscopy
Phase contrast and fluorescence images of OVCAR8-, COV318- and FT190-mCherry-eGFP-LC3B cells were captured using a Leica DMI 4000B inverted fluorescence microscope. Fluorescent images were captured using GFP and Y3 filter cubes and merged images were generated using the Leica Application Suite (LAS). Alternatively, STO-609-treated OVCAR8 mCherry-eGFP-LC3B cells were seeded at 2000 cells/well in 96-well round-bottom ULA plates. Each well was imaged by phase contrast, GFP and RFP using the IncuCyte® ZOOM live-cell imaging system (Sartorius) at 3h intervals for a total of 14 days; time-lapse videos were generated using the IncuCyte® built-in software. All images and videos are presented in their original format with no adjustments in colour or exposure correction.
Fluorescence quantification
Using Image J (Version 2.0.0-rc-69/1.52p), a region-of-interest (ROI) was circumscribed around each OVCAR8-mCherry-eGFP-LC3B spheroid (siNT- and siPRKAA1/2-transfected cells) using the phase contrast image as a template. The ROI was subsequently superimposed onto both the GFP and Y3 channel images where overall fluorescence intensity was measured in arbitrary units relative to overall spheroid area. Alternatively, GFP and RFP fluorescence, and signal overlap, were quantified on IncuCyte® ZOOM images of individual OVCAR8-mCherry-eGFP-LC3Bspheroids (n = 10) using the built-in analysis software and measured from days 2 to 12 as masked area per image.
Cell viability
Spheroids were collected by centrifugation at 2400 rpm for 3 min, washed with PBS, and centrifuged at 1000 rpm for 3 min. Pelleted spheroids were disaggregated using 50 μL of 0.25% Trypsin-EDTA incubated for 10 min at 37 °C followed by the addition of 50 μL FBS. Trypan Blue Reagent (Gibco) was added at a 1:1 ratio and cell counts were performed on the BioRad TC10™ automated cell counter.
Statistical analysis
Graphs were generated using GraphPad Prism 8 (La Jolla, California) and data are expressed as mean ± SD. Student’s t-test and ANOVA with either Dunnett’s or Sidak’s multiple comparison test were performed using GraphPad Prism 8; all results were considered significant at p < 0.05.
Results
Coordinated AMPK activity and LC3-II processing during spheroid formation
We demonstrated previously that AMPK is activated in EOC spheroids to promote cytostasis [8]. In an independent study, our group generated evidence that autophagy is rapidly induced in EOC spheroids also, and autophagy is required to maintain cell viability in these structures. Thus, we now seek to connect the kinetics and requirement for AMPK signaling with autophagy activation in our in vitro spheroid model of EOC metastasis. Assessment of autophagic flux can be initially performed by measuring protein expression of both microtubule-associated protein 1A/1B-light chain (LC3) and p62 (sequestosome-1). Being recruited to autophagosomal membranes, LC3 is proteolytically cleaved at its C-terminus followed by lipidation to generate LC3-II, making it an excellent marker for monitoring the progression of autophagy [12]. Due to its ubiquitin-binding domain, p62 is known to function as a mediator protein, targeting ubiquitinated proteins to the autophagosomal membrane. Accumulation of p62 protein levels is indicative of reduced autophagic flux, whereas its decrease over time indicates sustained autophagy induction [12].To address AMPK phosphorylation kinetics and its relation to autophagy induction, iOvCa147-MA cells were seeded in ULA conditions and protein was isolated at various time points during spheroid formation. Following immunoblot analysis, we identified increased levels of LC3-II processing and increased phosphorylation of AMPK at T172 between 24h and 72 h relative to adherent cells (Fig. 1a&b). The highest levels of both p-AMPK and LC3-II was observed at 48 h; therefore, subsequent spheroid culture experiments were taken to the 48 h time point. Time course experiments conducted using OVCAR8 spheroids further confirmed the 48 h time point as optimal for evaluating AMPK activity and autophagy markers (Fig. 1c&d). Different levels of basal autophagy were observed in standard adherent conditions between the iOvCa147-MA and OVCAR8 cell lines, as we have seen previously among several ovarian cancer cell lines [11].
Fig. 1
HGSOC spheroids have increased phosphorylated AMPK and LC3-II as compared with adherent cells. a iOvCa147-MA cells were trypsinized, seeded into ULA plates, and protein lysates were isolated at each time point as indicated. Adherent cell controls (adh) were cultured using standard tissue culture-treated plates for 72 h prior to protein isolation. Immunoblot analysis was performed for p-AMPK (T172), AMPK, and LC3B; tubulin served as a loading control. b Densitometric analysis of p-AMPK/AMPK and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 3) (*, p < 0.05). c OVCAR8 cells were trypsinized, seeded into ULA plates, and protein lysates were isolated at each time point as indicated. Adherent cell controls (adh) were cultured using standard tissue culture-treated plates for 72 h prior to protein isolation. Immunoblot analysis was performed for p-AMPK (T172), AMPK, and LC3B; tubulin served as a loading control. d Densitometric analysis of p-AMPK/AMPK and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 3) (*, p < 0.05)
HGSOC spheroids have increased phosphorylated AMPK and LC3-II as compared with adherent cells. a iOvCa147-MA cells were trypsinized, seeded into ULA plates, and protein lysates were isolated at each time point as indicated. Adherent cell controls (adh) were cultured using standard tissue culture-treated plates for 72 h prior to protein isolation. Immunoblot analysis was performed for p-AMPK (T172), AMPK, and LC3B; tubulin served as a loading control. b Densitometric analysis of p-AMPK/AMPK and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 3) (*, p < 0.05). c OVCAR8 cells were trypsinized, seeded into ULA plates, and protein lysates were isolated at each time point as indicated. Adherent cell controls (adh) were cultured using standard tissue culture-treated plates for 72 h prior to protein isolation. Immunoblot analysis was performed for p-AMPK (T172), AMPK, and LC3B; tubulin served as a loading control. d Densitometric analysis of p-AMPK/AMPK and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 3) (*, p < 0.05)
AMPK knockdown inhibits autophagic flux in EOC spheroids but does not alter p62 or LC3 processing
To elucidate the requirement of AMPK signaling regulation of autophagy in spheroids, we performed siRNA-mediated knockdown of the AMPK α1 and α2 catalytic subunits in iOvCa147-MA and OVCAR8 cells. AMPK exists as a heterotrimeric protein consisting of one catalytic α-subunit and two regulatory β- and γ-subunits. Although up to 12 different isomeric configurations are possible, there are only two known catalytic subunits encoded by the genes PRKAA1 and PRKAA2 [9]. Combined knockdown of PRKAA1 and PRKAA2 allowed us to control for variations in catalytic subunit expression and potential compensatory mechanisms, and to maximize AMPK attenuation. Following transfection in adherent conditions, cells were trypsinized and seeded into ULA conditions for 48 h, at which point protein was collected for immunoblot analysis. To our surprise, PRKAA1/2 knockdown in iOvCa147-MA or OVCAR8 spheroids did not significantly alter LC3-II or p62 relative to siNT-transfected control spheroids (Fig. 2a&b). This was intriguing since AMPK has been implicated in several models as a canonical activator of autophagy, with its loss typically inhibiting autophagic flux [14, 19, 20]. No significant difference in spheroid cell viability was observed between the PRKAA1/2 knockdown and siNT controls (data not shown), which corroborates the results from our previous study [8].
Fig. 2
PRKAA1/2 knockdown does not alter LC3-II and p62 levels in spheroids yet blocks autophagic flux. a Double knockdown of both AMPK α1 and α2 catalytic subunits was performed by co-transfection of PRKAA1 and PRKAA2 siRNA in adherent iOvCa147-MA and OVCAR8 cells; non-targeting siRNA (siNT) served as a control. At 72 h post-transfection, cells were trypsinized and seeded into 6-well ULA plates for 48 h. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for AMPK/tubulin, p62/tubulin, and LC3-II:I ratio from the immunoblots were tested for significance using a Student’s t-test (****, p < 0.001). c OVCAR8 mCherry-eGFP-LC3B cells were transfected with siNT and siPRKAA1/2 as described above and seeded into 24-well ULA plates. Phase contrast and fluorescence images were captured at 48 h post-seeding. Scale bar = 200 μm. d Quantification of eGFP (green markers) and mCherry (red markers) fluorescence intensity per spheroid (normalized to spheroid area) in siNT and siPRKAA1/2-transfected OVCAR8-mCherry-eGFP-LC3B cells was performed using Image J software and tested for significance by two-way ANOVA followed by Sidak’s multiple comparison test (**, p < 0.01; ****, p < 0.001)
PRKAA1/2 knockdown does not alter LC3-II and p62 levels in spheroids yet blocks autophagic flux. a Double knockdown of both AMPK α1 and α2 catalytic subunits was performed by co-transfection of PRKAA1 and PRKAA2 siRNA in adherent iOvCa147-MA and OVCAR8 cells; non-targeting siRNA (siNT) served as a control. At 72 h post-transfection, cells were trypsinized and seeded into 6-well ULA plates for 48 h. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for AMPK/tubulin, p62/tubulin, and LC3-II:I ratio from the immunoblots were tested for significance using a Student’s t-test (****, p < 0.001). c OVCAR8 mCherry-eGFP-LC3B cells were transfected with siNT and siPRKAA1/2 as described above and seeded into 24-well ULA plates. Phase contrast and fluorescence images were captured at 48 h post-seeding. Scale bar = 200 μm. d Quantification of eGFP (green markers) and mCherry (red markers) fluorescence intensity per spheroid (normalized to spheroid area) in siNT and siPRKAA1/2-transfected OVCAR8-mCherry-eGFP-LC3B cells was performed using Image J software and tested for significance by two-way ANOVA followed by Sidak’s multiple comparison test (**, p < 0.01; ****, p < 0.001)To further investigate the effect of PRKAA1/2 knockdown on autophagic flux in EOC spheroids, we used OVCAR8 cells stably-transfected with an eGFP-LC3B reporter construct [10]. Following PRKAA1/2 knockdown, OVCAR8-eGFP-LC3B cells were seeded as spheroids and assessed using live-cell fluorescence microscopy. We observed a notable increase in green fluorescence in spheroids following PRKAA1/2 knockdown indicating a block in autophagic flux (Figure S1). However, it is difficult to draw this conclusion, as well as adequately monitor autophagic progression from early-to-late stages, with a single fluorescence reporter construct. To address this issue, we stably transfected OVCAR8 cells with the dual fluorescence mCherry-eGFP-LC3B reporter [21]. Following autophagosome fusion with the acidic lysosome, the pH-sensitive eGFP signal is quenched, whereas the mCherry signal remains unaffected. Highly autophagic cells will exhibit predominantly red fluorescent punctae indicative of increased autophagic flux. Conversely, inhibiting autophagy induces an increase in green fluorescence due to reduced autophagosome fusion with lysosomes. Although this reporter has been used in adherent culture systems [21, 22], it can also be applied to spheroid models [23]. By placing OVCAR8 mCherry-eGFP-LC3B cells into ULA conditions and assessing overall fluorescence colour shift rather than individual autophagic punctae, we can characterize general autophagic flux within spheroids in a rapid manner.PRKAA1/2 knockdown in OVCAR8 mCherry-eGFP-LC3Bspheroids resulted in a dramatic increase in green and red fluorescence relative to siNT-transfected control spheroids, which had predominantly low levels of fluorescence signal (Fig. 2c&d). To confirm our interpretation of a block in autophagic flux, we treated spheroids with chloroquine (CQ), a well-characterized lysosomotropic agent that inhibits lysosomal fusion to the autophagosome [12], and which we have demonstrated previously inhibits autophagy in EOC cells and spheroids [10, 11]. Treatment of OVCAR8 mCherry-eGFP-LC3Bspheroids with 50 μM CQ for 4 h resulted in similar accumulation of green fluorescence as we observed with the PRKAA1/2 knockdown (data not shown). Thus, PRKAA1/2 knockdown can reduce autophagic flux in EOC spheroids; however, based on our immunoblot data, this observed AMPK-mediated regulation of autophagy may occur in an LC3- and p62-independent manner.In addition to PRKAA1/2 knockdown, we sought to examine the effect of a pharmacological inhibitor of AMPK on EOC spheroids. Currently, Compound C (also known as dorsomorphin) is the only known selective inhibitor of AMPK [24]. Treatment of both iOvCa147-MA and OVCAR8 cells with Compound C resulted in modest reduction of p-AMPK at 10 μM (Fig. 3a), yet significant increases in LC3 processing and a slight increase in p62 levels were observed (Fig. 3a&b). OVCAR8 mCherrry-eGFP-LC3Bspheroids treated with 10 μM Compound C for 24 h exhibited a detectable increase in green fluorescence relative to their DMSO-treated controls (Fig. 3c).
Fig. 3
Pharmacologic inhibition of AMPK using Compound C increases LC3-II and blocks autophagic flux in spheroids. a iOvCa147-MA and OVCAR8 cells were seeded into 6-well ULA plates to form spheroids for 24 h prior to treatment with Compound C at the indicated concentrations; DMSO was the vehicle control. Protein lysates were isolated at 24 h post-treatment. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for p62/tubulin and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test. Asterisks indicate significant differences relative to control (*, p < 0.05; **, p < 0.01). c OVCAR8 mCherry-eGFP-LC3B cells were seeded into 24-well ULA plates, cultured for 24 h, then treated with 10 μM Compound C, or DMSO as a control, for an additional 24 h. Phase contrast and fluorescence images were captured. Scale bar = 200 μm
Pharmacologic inhibition of AMPK using Compound C increases LC3-II and blocks autophagic flux in spheroids. a iOvCa147-MA and OVCAR8 cells were seeded into 6-well ULA plates to form spheroids for 24 h prior to treatment with Compound C at the indicated concentrations; DMSO was the vehicle control. Protein lysates were isolated at 24 h post-treatment. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for p62/tubulin and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test. Asterisks indicate significant differences relative to control (*, p < 0.05; **, p < 0.01). c OVCAR8 mCherry-eGFP-LC3B cells were seeded into 24-well ULA plates, cultured for 24 h, then treated with 10 μM Compound C, or DMSO as a control, for an additional 24 h. Phase contrast and fluorescence images were captured. Scale bar = 200 μm
AMPK activation alone is insufficient to induce autophagy
We have shown previously that proliferating adherent EOC cells have relatively low levels of both autophagy and p-AMPK yet are robustly induced upon spheroid formation [8, 11]. As such, we deemed it important to test whether AMPK activity on its own is sufficient to induce autophagy in EOC cells. To achieve this, we activated AMPK by using the mitochondrial inhibitors oligomycin (100 nM) and metformin (2 mM), since both drugs are known to increase p-AMPK and its activity [25]. Oligomycin and metformin treatment for 24 h led to increased p-AMPK in adherent OVCAR8 cells, however no significant changes were observed in LC3-II processing or p62 levels (Figure S2). Taken together, our results suggest that AMPK activation is required in part for autophagic flux in EOC spheroids, yet on its own is insufficient to induce autophagy in adherent EOC cells.
Pharmacologic inhibition of CAMKKβ reduces AMPK phosphorylation and inhibits autophagic flux
Due to the limited availability of small molecule inhibitors of AMPK, we sought to attenuate AMPK phosphorylation by targeting upstream kinases that lead to AMPK activation. Liver Kinase B1 (LKB1) encoded by STK11, is the best-characterized upstream kinase of AMPK. LKB1 is a highly-conserved serine-threonine kinase that typically functions as a regulator of cellular metabolism within the AMPK signaling axis [26]. Surprisingly, recent work from our laboratory identified that EOC spheroids lacking LKB1 expression by CRISPR-mediated STK11 knockout sustain elevated p-AMPK suggesting alternative kinase(s) target AMPK in our system [27].Previous literature has implicated calcium/calmodulin-dependent protein kinase beta (CAMKKβ) as an alternative AMPK activating kinase [28]. For example, cellular matrix deprivation leads to CAMKKβ-mediated AMPK phosphorylation in breast cancer cell lines [29]. As such, we decided to use a selective CAMKKβ inhibitor, STO-609, as another method to attenuate AMPK phosphorylation. We included additional cell lines, the high-grade serous cancerCOV318 cells, and immortalized human fallopian tube secretory epithelial FT190 cells. Treatment of non-malignant FT190 and EOC cell line spheroids with 10 μM ST0–609 resulted in significant reduction in p-AMPK (Fig. 4a). In addition, we observed a significant increase in p62, but no change in LC3-II levels (Fig. 4b). ST0–609 treated spheroids exhibited increased green fluorescence relative to their DMSO control indicating robust autophagic flux inhibition (Fig. 4c). Enhanced green fluorescence was observed in FT190 spheroids, suggesting that CAMKKβ-mediated regulation of AMPK and autophagic flux occur in both EOC cells and their potential premalignant precursor cells, too.
Fig. 4
STO-609 treatment reduces p-AMPK, increases p62, and blocks autophagic flux in spheroids. a Cell lines (EOC cells: iOvCa147-MA, OVCAR8 and COV318; non-malignant cells: FT190) were seeded into 6-well ULA plates to form spheroids for 24 h prior to treatment with 10 μM STO-609, or DMSO as a vehicle control. Protein lysates were isolated at 24 h post-treatment. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for p-AMPK/AMPK, p62/tubulin and LC3-II:I ratio from the immunoblots for the three EOC cell lines together were tested by Student’s t-test. Asterisks indicate significant differences relative to control (*, p < 0.05; ****, p < 0.001). c Representative phase contrast and fluorescence images of DMSO- and STO-609-treated spheroids using OVCAR8-mCherry-eGFP-LC3B, COV318-mCherry-eGFP-LC3B, and FT190-mCherry-eGFP-LC3B cells. Treatments were performed as described above and images were captured after 24 h. Scale bar = 200 μm
STO-609 treatment reduces p-AMPK, increases p62, and blocks autophagic flux in spheroids. a Cell lines (EOC cells: iOvCa147-MA, OVCAR8 and COV318; non-malignant cells: FT190) were seeded into 6-well ULA plates to form spheroids for 24 h prior to treatment with 10 μM STO-609, or DMSO as a vehicle control. Protein lysates were isolated at 24 h post-treatment. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for p-AMPK/AMPK, p62/tubulin and LC3-II:I ratio from the immunoblots for the three EOC cell lines together were tested by Student’s t-test. Asterisks indicate significant differences relative to control (*, p < 0.05; ****, p < 0.001). c Representative phase contrast and fluorescence images of DMSO- and STO-609-treated spheroids using OVCAR8-mCherry-eGFP-LC3B, COV318-mCherry-eGFP-LC3B, and FT190-mCherry-eGFP-LC3B cells. Treatments were performed as described above and images were captured after 24 h. Scale bar = 200 μmWe have demonstrated previously that autophagy is critical to maintain EOC cell viability in spheroids [10, 11], thus we postulate that potent inhibition of AMPK activity using STO-609 over an extended period would negatively impact EOC spheroid cell viability. First, we treated OVCAR8 mCherrry-eGFP-LC3Bspheroids with 10 μM STO-609 or DMSO for up to 12 days to visualize extent of autophagic flux inhibition and spheroid integrity. Autophagic flux was potently blocked by STO-609 over the complete time period as evidenced by increased green fluorescence; we also observed a small decrease in overall spheroid size due to STO-609 treatment (Fig. 5a&b). Subsequently, we evaluated the effects of CAMKKβ-AMPK signaling inhibition by treating nine different high-grade serous EOC spheroids, and FT190 spheroid controls, with STO-609 for 3 and 7 days prior to quantifying viable cell number. After 3 days of STO-609 treatment, we observed a significant reduction in cell viability in 6 out of 9 EOC cell line spheroids (Fig. 6a); extending this treatment to 7 days resulted in an additional cell line (OVCAR4) sensitive to CAMKKβ-AMPK inhibition (Fig. 6b). Cell viability for two EOC cell lines, CaOV3 and COV318, as well as normal FT190 spheroids, was unaffected by STO-609 treatment at both time points. In summary, our results implicate CAMKKβ-mediated activation of AMPK is required for autophagy induction and resultant cell survival in EOC spheroids.
Fig. 5
Time course of STO-609-mediated inhibition of autophagic flux in OVCAR8 mCherry-eGFP-LC3B spheroids. a OVCAR8 mCherry-eGFP-LC3B cells were pretreated with either 10 μM STO-609 or DMSO and seeded at a density of 2000 cells/well into 96-well round-bottom ULA plates. Images of eGFP and mCherry fluorescence signals were captured using the IncuCyte® ZOOM live-cell imaging system at 3h intervals over a period of 14 days. Representative images are shown for specific time points (days 2–12). Scale bar = 200 μm. b Quantification of eGFP, mCherry and Overlap fluorescence signals were quantified from 10 independent wells using the IncuCyte® ZOOM image analysis software. Data (mean ± SD) are displayed starting at day 2 to allow for complete aggregation of cells into individual spheroids, and to avoid the increased background fluorescence signal on image edges in the GFP channel at these early time points
Fig. 6
STO-609 treatment reduces spheroid viability across several EOC cell line spheroids. Cells were seeded into 24-well ULA plates and treated with 10 μM STO-609 or DMSO at the time of seeding. Trypan Blue Exclusion cell counting was performed at days 3 (a) and 6 (b); viability data was normalized to DMSO-treated controls set to 100%. Student’s t-test was performed to determine statistical significance for each cell line (*, p < 0.05)
Time course of STO-609-mediated inhibition of autophagic flux in OVCAR8 mCherry-eGFP-LC3Bspheroids. a OVCAR8 mCherry-eGFP-LC3B cells were pretreated with either 10 μM STO-609 or DMSO and seeded at a density of 2000 cells/well into 96-well round-bottom ULA plates. Images of eGFP and mCherry fluorescence signals were captured using the IncuCyte® ZOOM live-cell imaging system at 3h intervals over a period of 14 days. Representative images are shown for specific time points (days 2–12). Scale bar = 200 μm. b Quantification of eGFP, mCherry and Overlap fluorescence signals were quantified from 10 independent wells using the IncuCyte® ZOOM image analysis software. Data (mean ± SD) are displayed starting at day 2 to allow for complete aggregation of cells into individual spheroids, and to avoid the increased background fluorescence signal on image edges in the GFP channel at these early time pointsSTO-609 treatment reduces spheroid viability across several EOC cell line spheroids. Cells were seeded into 24-well ULA plates and treated with 10 μM STO-609 or DMSO at the time of seeding. Trypan Blue Exclusion cell counting was performed at days 3 (a) and 6 (b); viability data was normalized to DMSO-treated controls set to 100%. Student’s t-test was performed to determine statistical significance for each cell line (*, p < 0.05)
Discussion
Dysregulation of autophagy has long been implicated in numerous pathologies [30]. In the context of metastatic ovarian cancer, it appears that autophagy serves a tumour protective role. We have demonstrated previously that EOC spheroids display increased levels of autophagy and that its inhibition can reduce overall EOC spheroid viability [10, 11]. Independently, we demonstrated that p-AMPK is increased in both patient-derived EOC spheroids as well as those generated in vitro, relative to proliferating adherent cells [8]. In this study, we sought to bridge these two phenomena and demonstrate that AMPK activity is required for autophagy induction in EOC spheroids to maintain cell viability.In several biological contexts, AMPK activity on its own can lead directly to autophagy induction. However, treatment of adherent OVCAR8 cells with AMPK activators did not significantly change LC3-II or p62 expression. Metformin and oligomycin treatments increased p-AMPK levels, but these drugs function indirectly to activate AMPK by inhibiting mitochondrial respiration [31, 32]. Thus, caution must be taken when using these methods for AMPK activation and correlating results with autophagy induction. However, we consider it unlikely that AMPK activation is sufficient on its own to induce autophagy in proliferating adherent EOC cells.We show that RNAi-mediated AMPK inhibition strongly inhibits autophagic flux as visualized by fluorescence microscopy. Interestingly, this phenotype may occur in an LC3- and p62-independent manner. The discrepancy in our results between fluorescence reporter and immunoblot assays raise certain questions as to what specific players mediate autophagy induction in ovarian cancer. Initially described in yeast as ATG8, several orthologs of the ubiquitin-like LC3 protein have been identified in mammals, although most work focuses on LC3B. More recently, studies have implicated LC3B-independent forms of autophagy. One candidate LC3 ortholog is gamma-aminobutyric acid receptor-associated protein (GABARAP). GABARAP has been shown to possess separate functions from LC3, as it is involved in late-stage autophagosome maturation [33]. More recently, LC3-independent autophagy in rat hepatocytes was shown to be regulated primarily through the GABARAP complex in the autophagosome [34]. Since we did not observe major effects on LC3 processing, we are currently investigating whether AMPK inhibition affects GABARAP expression and function in autophagic flux in EOC spheroids.Although Compound C did not attenuate p-AMPK levels nearly to the same extent as either RNA interference or STO-609 treatment, it clearly inhibited autophagic flux in EOC spheroids. Compound C may affect autophagy as a combination of AMPK inhibition as well as with other potential targets of this agent. Previous literature identified multiple intersecting pathways that are potently affected by Compound C that are independent of AMPK (Harhaji-Trajkovic et al., 2010; Zhao et al., 2018). In fact, Compound C is known to affect BMP and mTOR signaling [24, 35]; we have demonstrated that both of these signaling pathways impinge upon the EOC spheroid phenotype [7, 10, 36]. As such, the combined action of Compound C on multiple different kinases could lead to our observed LC3 reporter results in EOC spheroids. In fact, we observed poor p-AMPK attenuation using Compound C, and it has conflicting roles as either an activator or inhibitor of autophagy [35]. Thus, use of this agent alone poses a limitation for analysis of AMPK regulation of autophagy in our system.To address this, we present new findings regarding the requirement of CAMKKβ-mediated AMPK signaling in modulating autophagy in EOC. Treatment of EOC spheroids with the CAMKKβ inhibitor, STO-609, supports our PRKAA1/2 knockdown data, thus strengthening the notion that AMPK is required for autophagy induction in EOC cells under spheroid conditions. This phenotype holds true not only for EOC cell lines, but also in non-malignant fallopian tube epithelial cells. Perhaps the autophagic stress response mediated by AMPK is conserved in secretory epithelial cells, as well as the high-grade serous EOC cells from which they arise.Furthermore, work in our laboratory identified recently that LKB1-deficient EOC spheroids still retain the capacity to induce p-AMPK through CAMKKβ activity [27]. This finding together with our results herein suggest a crucial role for CAMKKβ in regulating p-AMPK levels in this disease. It has been previously reported that a rise in cytosolic calcium can induce autophagy through CAMKKβ in both MCF-7 and HeLa cell lines, highlighting an ATP-independent mechanism for autophagy induction [37]. More recently, cellular matrix deprivation has been identified as an inducer of intracellular calcium spikes, which in turn can activate AMPK through CAMKKβ signaling [29]. Examination of the calcium-oxidant signaling network in EOC spheroids might highlight a unique characteristic of these cancer cells that would lend itself to therapeutic inhibition. As such, it would be prudent to further characterize both the AMPK-dependent and -independent roles of CAMKKβ in the context of ovarian cancer. Our encouraging results of CAMKKβ-AMPK inhibition using the STO-609 and its negative impact on EOC spheroid cell viability lends even more support for such an intervention.Overall, it appears that AMPK is required in part to induce autophagy in EOC spheroids, although this may occur in an LC3-and p62-independent manner. We also show AMPK phosphorylation is regulated by CAMKKβ activity in EOC spheroids to promote autophagic flux in these structures. These findings have contributed to our understanding of signaling axes regulating autophagy induction in EOC cells, and may represent novel therapeutic targets for this critical stress response in the setting of metastatic disease.Additional file 1: Figure S1.PRKAA1/2 knockdown potentially inhibits autophagy in OVCAR8 eGFP-LC3Bspheroids. Adherent cells were transfected with non-targeting siRNA (siNT) or siPRKAA1/2, or left untransfected, for 72 h. Cells were seeded into 24-well ULA culture dishes for 48 h prior to capturing phase contrast and fluorescence images. Scale bar = 200 μm.Additional file 2: Figure S2. Pharmacologic AMPK activation does not alter LC3 processing and p62 levels in adherent iOvCa147-MA and OVCAR8 cells. (a) iOvCa147-MA and OVCAR8 cells were plated at a density of 150,000 cells/well in 6-well tissue-culture-treated plates and left to attach overnight. Cells were subsequently treated for 24 h with either Oligomycin (100 nM), or Metformin (2 mM, iOvCa147-MA; 1 mM, OVCAR8), or DMSO vehicle control. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62 and LC3B; tubulin served as a loading control. (b) Densitometric analysis of p62/tubulin and LC3-II:I ratio from the immunoblots were tested by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 3) and no significant differences were observed.
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Hua Feng; Yibin Feng; Yuchen Feng; Thomas A Ferguson; Álvaro F Fernández; Maite G Fernandez-Barrena; Jose C Fernandez-Checa; Arsenio Fernández-López; Martin E Fernandez-Zapico; Olivier Feron; Elisabetta Ferraro; Carmen Veríssima Ferreira-Halder; Laszlo Fesus; Ralph Feuer; Fabienne C Fiesel; Eduardo C Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; John H Fingert; Steven Finkbeiner; Toren Finkel; Filomena Fiorito; Paul B Fisher; Marc Flajolet; Flavio Flamigni; Oliver Florey; Salvatore Florio; R Andres Floto; Marco Folini; Carlo Follo; Edward A Fon; Francesco Fornai; Franco Fortunato; Alessandro Fraldi; Rodrigo Franco; Arnaud Francois; Aurélie François; Lisa B Frankel; Iain Dc Fraser; Norbert Frey; Damien G Freyssenet; Christian Frezza; Scott L Friedman; Daniel E Frigo; Dongxu Fu; José M Fuentes; Juan Fueyo; Yoshio Fujitani; Yuuki Fujiwara; Mikihiro Fujiya; Mitsunori Fukuda; Simone Fulda; Carmela Fusco; Bozena Gabryel; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Sehamuddin Galadari; Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; Nelly Godefroy; Robert M Gogal; Kuppan Gokulan; Gustavo H Goldman; Delia Goletti; Michael S Goligorsky; Aldrin V Gomes; Ligia C Gomes; Hernando Gomez; Candelaria Gomez-Manzano; Rubén Gómez-Sánchez; Dawit Ap Gonçalves; Ebru Goncu; Qingqiu Gong; Céline Gongora; Carlos B Gonzalez; Pedro Gonzalez-Alegre; Pilar Gonzalez-Cabo; Rosa Ana González-Polo; Ing Swie Goping; Carlos Gorbea; Nikolai V Gorbunov; Daphne R Goring; Adrienne M Gorman; Sharon M Gorski; Sandro Goruppi; Shino Goto-Yamada; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Yacine Graba; Martin Graef; Giovanna E Granato; Gary Dean Grant; Steven Grant; Giovanni Luca Gravina; Douglas R Green; Alexander Greenhough; Michael T Greenwood; Benedetto Grimaldi; Frédéric Gros; Charles Grose; Jean-Francois Groulx; Florian Gruber; Paolo Grumati; Tilman Grune; Jun-Lin Guan; Kun-Liang Guan; Barbara Guerra; Carlos Guillen; Kailash Gulshan; Jan Gunst; Chuanyong Guo; Lei Guo; Ming Guo; Wenjie Guo; Xu-Guang Guo; Andrea A Gust; Åsa B Gustafsson; Elaine Gutierrez; Maximiliano G Gutierrez; Ho-Shin Gwak; Albert Haas; James E Haber; Shinji Hadano; Monica Hagedorn; David R Hahn; Andrew J Halayko; Anne Hamacher-Brady; Kozo Hamada; Ahmed Hamai; Andrea Hamann; Maho Hamasaki; Isabelle Hamer; Qutayba Hamid; Ester M Hammond; Feng Han; Weidong Han; James T Handa; John A Hanover; Malene Hansen; Masaru Harada; Ljubica Harhaji-Trajkovic; J Wade Harper; Abdel Halim Harrath; Adrian L Harris; James Harris; Udo Hasler; Peter Hasselblatt; Kazuhisa Hasui; Robert G Hawley; Teresa S Hawley; Congcong He; Cynthia Y He; Fengtian He; Gu He; Rong-Rong He; Xian-Hui He; You-Wen He; Yu-Ying He; Joan K Heath; Marie-Josée Hébert; Robert A Heinzen; Gudmundur Vignir Helgason; Michael Hensel; Elizabeth P Henske; Chengtao Her; Paul K Herman; Agustín Hernández; Carlos Hernandez; Sonia Hernández-Tiedra; Claudio Hetz; P Robin Hiesinger; Katsumi Higaki; Sabine Hilfiker; Bradford G Hill; Joseph A Hill; William D Hill; Keisuke Hino; Daniel Hofius; Paul Hofman; Günter U Höglinger; Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; Rossella Menghini; A Sue Menko; Rubem Fs Menna-Barreto; Manoj B Menon; Marco A Meraz-Ríos; Giuseppe Merla; Luciano Merlini; Angelica M Merlot; Andreas Meryk; Stefania Meschini; Joel N Meyer; Man-Tian Mi; Chao-Yu Miao; Lucia Micale; Simon Michaeli; Carine Michiels; Anna Rita Migliaccio; Anastasia Susie Mihailidou; Dalibor Mijaljica; Katsuhiko Mikoshiba; Enrico Milan; Leonor Miller-Fleming; Gordon B Mills; Ian G Mills; Georgia Minakaki; Berge A Minassian; Xiu-Fen Ming; Farida Minibayeva; Elena A Minina; Justine D Mintern; Saverio Minucci; Antonio Miranda-Vizuete; Claire H Mitchell; Shigeki Miyamoto; Keisuke Miyazawa; Noboru Mizushima; Katarzyna Mnich; Baharia Mograbi; Simin Mohseni; Luis Ferreira Moita; Marco Molinari; Maurizio Molinari; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Marco Mongillo; Martha M Monick; Serena Montagnaro; Craig Montell; Darren J Moore; Michael N Moore; Rodrigo Mora-Rodriguez; Paula I Moreira; Etienne Morel; Maria Beatrice Morelli; Sandra Moreno; Michael J Morgan; Arnaud Moris; Yuji Moriyasu; Janna L Morrison; Lynda A Morrison; Eugenia Morselli; Jorge Moscat; Pope L Moseley; Serge Mostowy; Elisa Motori; Denis Mottet; Jeremy C Mottram; Charbel E-H Moussa; Vassiliki E Mpakou; Hasan Mukhtar; Jean M Mulcahy Levy; Sylviane Muller; Raquel Muñoz-Moreno; Cristina Muñoz-Pinedo; Christian Münz; Maureen E Murphy; James T Murray; Aditya Murthy; Indira U Mysorekar; Ivan R Nabi; Massimo Nabissi; Gustavo A Nader; Yukitoshi Nagahara; Yoshitaka Nagai; Kazuhiro Nagata; Anika Nagelkerke; Péter Nagy; Samisubbu R Naidu; Sreejayan Nair; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Gennaro Napolitano; Naweed I Naqvi; Roberta Nardacci; Derek P Narendra; Masashi Narita; Anna Chiara Nascimbeni; Ramesh Natarajan; Luiz C Navegantes; Steffan T Nawrocki; Taras Y Nazarko; Volodymyr Y Nazarko; Thomas Neill; Luca M Neri; Mihai G Netea; Romana T Netea-Maier; Bruno M Neves; Paul A Ney; Ioannis P Nezis; Hang Tt Nguyen; Huu Phuc Nguyen; Anne-Sophie Nicot; Hilde Nilsen; Per Nilsson; Mikio Nishimura; Ichizo Nishino; Mireia Niso-Santano; Hua Niu; Ralph A Nixon; Vincent Co Njar; Takeshi Noda; Angelika A Noegel; Elsie Magdalena Nolte; Erik Norberg; Koenraad K Norga; Sakineh Kazemi Noureini; Shoji Notomi; Lucia Notterpek; Karin Nowikovsky; Nobuyuki Nukina; Thorsten Nürnberger; Valerie B O'Donnell; Tracey O'Donovan; Peter J O'Dwyer; Ina Oehme; Clara L Oeste; Michinaga Ogawa; Besim Ogretmen; Yuji Ogura; Young J Oh; Masaki Ohmuraya; Takayuki Ohshima; Rani Ojha; Koji Okamoto; Toshiro Okazaki; F Javier Oliver; Karin Ollinger; Stefan Olsson; Daniel P Orban; Paulina Ordonez; Idil Orhon; Laszlo Orosz; Eyleen J O'Rourke; Helena Orozco; Angel L Ortega; Elena Ortona; Laura D Osellame; Junko Oshima; Shigeru Oshima; Heinz D Osiewacz; Takanobu Otomo; Kinya Otsu; Jing-Hsiung James Ou; Tiago F Outeiro; Dong-Yun Ouyang; Hongjiao Ouyang; Michael Overholtzer; Michelle A Ozbun; P Hande Ozdinler; Bulent Ozpolat; Consiglia Pacelli; Paolo Paganetti; Guylène Page; Gilles Pages; Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; Celine Perier; Andras Perl; David H Perlmutter; Ida Perrotta; Shazib Pervaiz; Maija Pesonen; Jeffrey E Pessin; Godefridus J Peters; Morten Petersen; Irina Petrache; Basil J Petrof; Goran Petrovski; James M Phang; Mauro Piacentini; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Federico Pietrocola; Felipe X Pimentel-Muiños; Mario Pinar; Benjamin Pineda; Ronit Pinkas-Kramarski; Marcello Pinti; Paolo Pinton; Bilal Piperdi; James M Piret; Leonidas C Platanias; Harald W Platta; Edward D Plowey; Stefanie Pöggeler; Marc Poirot; Peter Polčic; Angelo Poletti; Audrey H Poon; Hana Popelka; Blagovesta Popova; Izabela Poprawa; Shibu M Poulose; Joanna Poulton; Scott K Powers; Ted Powers; Mercedes Pozuelo-Rubio; Krisna Prak; Reinhild Prange; Mark Prescott; Muriel Priault; Sharon Prince; Richard L Proia; Tassula Proikas-Cezanne; Holger Prokisch; Vasilis J Promponas; Karin Przyklenk; Rosa Puertollano; Subbiah Pugazhenthi; Luigi Puglielli; Aurora Pujol; Julien Puyal; Dohun Pyeon; Xin Qi; Wen-Bin Qian; Zheng-Hong Qin; Yu Qiu; Ziwei Qu; Joe Quadrilatero; Frederick Quinn; Nina Raben; Hannah Rabinowich; Flavia Radogna; Michael J Ragusa; Mohamed Rahmani; Komal Raina; Sasanka Ramanadham; Rajagopal Ramesh; Abdelhaq Rami; Sarron Randall-Demllo; Felix Randow; Hai Rao; V Ashutosh Rao; Blake B Rasmussen; Tobias M Rasse; Edward A Ratovitski; Pierre-Emmanuel Rautou; Swapan K Ray; Babak Razani; Bruce H Reed; Fulvio Reggiori; Markus Rehm; Andreas S Reichert; Theo Rein; David J Reiner; Eric Reits; Jun Ren; Xingcong Ren; Maurizio Renna; Jane Eb Reusch; Jose L Revuelta; Leticia Reyes; Alireza R Rezaie; Robert I Richards; Des R Richardson; Clémence Richetta; Michael A Riehle; Bertrand H Rihn; Yasuko Rikihisa; Brigit E Riley; Gerald Rimbach; Maria Rita Rippo; Konstantinos Ritis; Federica Rizzi; Elizete Rizzo; Peter J Roach; Jeffrey Robbins; Michel Roberge; Gabriela Roca; Maria Carmela Roccheri; Sonia Rocha; Cecilia Mp Rodrigues; Clara I Rodríguez; Santiago Rodriguez de Cordoba; Natalia Rodriguez-Muela; Jeroen Roelofs; Vladimir V Rogov; Troy T Rohn; Bärbel Rohrer; Davide Romanelli; Luigina Romani; Patricia Silvia Romano; M Isabel G Roncero; Jose Luis Rosa; Alicia Rosello; Kirill V Rosen; Philip Rosenstiel; Magdalena Rost-Roszkowska; Kevin A Roth; Gael Roué; Mustapha Rouis; Kasper M Rouschop; Daniel T Ruan; Diego Ruano; David C Rubinsztein; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Ruediger Rudolf; Markus A Ruegg; Carmen Ruiz-Roldan; Avnika Ashok Ruparelia; Paola Rusmini; David W Russ; Gian Luigi Russo; Giuseppe Russo; Rossella Russo; Tor Erik Rusten; Victoria Ryabovol; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Michael Sacher; Carsten Sachse; Michael N Sack; Junichi Sadoshima; Paul Saftig; Ronit Sagi-Eisenberg; Sumit Sahni; Pothana Saikumar; Tsunenori Saito; Tatsuya Saitoh; Koichi Sakakura; Machiko Sakoh-Nakatogawa; Yasuhito Sakuraba; María Salazar-Roa; Paolo Salomoni; Ashok K Saluja; Paul M Salvaterra; Rosa Salvioli; Afshin Samali; Anthony Mj Sanchez; José A Sánchez-Alcázar; Ricardo Sanchez-Prieto; Marco Sandri; Miguel A Sanjuan; Stefano Santaguida; Laura Santambrogio; Giorgio Santoni; Claudia Nunes Dos Santos; Shweta Saran; Marco Sardiello; Graeme Sargent; Pallabi Sarkar; Sovan Sarkar; Maria Rosa Sarrias; Minnie M Sarwal; Chihiro Sasakawa; Motoko Sasaki; Miklos Sass; Ken Sato; Miyuki Sato; Joseph Satriano; Niramol Savaraj; Svetlana Saveljeva; Liliana Schaefer; Ulrich E Schaible; Michael Scharl; Hermann M Schatzl; Randy Schekman; Wiep Scheper; Alfonso Schiavi; Hyman M Schipper; Hana Schmeisser; Jens Schmidt; Ingo Schmitz; Bianca E Schneider; E Marion Schneider; Jaime L Schneider; Eric A Schon; Miriam J Schönenberger; Axel H Schönthal; Daniel F Schorderet; Bernd Schröder; Sebastian Schuck; Ryan J Schulze; Melanie Schwarten; Thomas L Schwarz; Sebastiano Sciarretta; Kathleen Scotto; A Ivana Scovassi; Robert A Screaton; Mark Screen; Hugo Seca; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Jose M Seguí-Simarro; Juan Segura-Aguilar; Ekihiro Seki; Christian Sell; Iban Seiliez; 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