Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Introduction

Na+,K+-ATPase is an integral plasma membrane protein consisting of α-, β- and γ-subunits. It has been shown that ATP hydrolysis leads to phosphorylation of residue Asp369 in the α-subunit, providing E1–E2 conformational transition and electrogenic ion transport (3 Na+ versus 2 K+) at a baseline rate of 60–80 phosphorylation-dephosphorylation cycles per second. In addition to the ubiquitous α1-subunit, three other α-subunits are expressed in a tissue-specific manner. The α2-subunit is mainly present in skeletal muscle, heart, and brain, the α3-subunit predominates in nervous tissue, and the α4-subunit was found in testis [1-4]. In nervous tissue, the α1-subunit is expressed in both neurons and glial cells, the α2-subunit is primarily found in astrocytes and oligodendrocytes, whereas neurons are abundant in the α3-subunit [5]. Three β isoforms (β1, β2, β3) have been characterized in mammalian cells [6]. The β isoforms also exhibit a tissue-specific pattern of expression. The β1-subunit is found in nearly every tissue, the β2 isoform is found in skeletal muscle, pineal gland, and nervous tissue, and β3 is present in lung, retina, liver, and testis [7]. The β-subunits are highly glycosylated and are obligatory for delivery, conformational stability, and enzymatic activity. The γ-subunit from the family of FXYD proteins (that share a Pro-Phe-X-Tyr-Asp motif) is involved in regulation of Na+/K+-ATPase by interacting with the α- and β-subunits. The expression of FXYD proteins is tissue-specific, for example, FXYD1, mainly expressed in heart and skeletal muscle, FXYD2 (γ-subunit) and FXYD4 expressed in renal tubule, FXYD3 expressed in stomach, colon, and numerous tumors, and FXYD7 is exclusively expressed in the brain in both neurons and glial cells. It has been shown that FXYD proteins affect the apparent affinity of Na+/K+-ATPase for extracellular K+ [8-11] and intracellular Na+ [12-14].

Cardiotonic steroids (CTSs) are known as compounds sharing a common structure formed by a steroid nucleus with a lactone ring at C-17 and a hydroxyl group at C-14. Lactone rings with 5 or 6 members are the most significant feature of cardenolides and bufadienolides, which are isolated from plants and amphibians, respectively. The mechanism of inhibition of Na+,K+-ATPase by CTSs has been explored mainly with ouabain extracted from Strophanthus gratus. As predicted, exposure to CTSs affects numerous cellular functions related to maintenance of the transmembrane gradient of Na+ and K+, such as electrical membrane potential (Em), cell volume, transepithelial movement of salt and osmotically obliged water, Na+/H+ and Na+(K+)/Ca2+ exchange, symport of Na+ with inorganic phosphate, glucose, amino acids, and nucleotides, etc. During the last two decades, it has been shown that along with the above-mentioned canonical [Na+]i,[K+]i-, Em-, and cell volume-mediated cellular responses, CTSs can affect gene expression, membrane trafficking, and cell adhesion, proliferation, and death. The data on relative impacts of [Na+]i,[K+]i-mediated and -independent signaling in these noncanonical cellular responses triggered by CTSs are controversial (for reviews, see [15-19]).

Unlike most other cells, pyramidal and dentate neurons of the cerebral cortex and hippocampus [20-21] as well as rat granule cell culture [22, 23] are abundant with the α3-Na+,K+-ATPase isoform. Using HeLa cells transfected with cDNAs encoding distinct catalytic subunits, Jewell and Lingrel found that apparent affinity for Na+ of the α1 and α2 isoforms is 2-3-fold higher compared to the α3 isoform [24]. In α1-α3-transfected, monensin-treated HeLa cells the apparent K0.5 values of ouabain-sensitive 86Rb influx for cytoplasmic Na+ activation were ~18, 20, and 64 mM for the α1, α2, and α3 isoforms, respectively [25]. The attenuated affinity of the α3 isoform for [Na+]i was also confirmed in a study of the kinetics of Na+ efflux from α1-α3-transfected HeLa cells loaded with Na+-sensitive fluorescent dye SBFI [26]. Keeping these differences in mind, it has been proposed that α1-Na+,K+-ATPase isoform plays a key role in the maintenance of low [Na+]i/[K+]i ratio under baseline conditions, whereas the α3- Na+,K+-ATPase isoform is involved in normalization of the gain of [Na+]i evoked by sustained neuronal excitation [26-28].

Numerous research teams have reported that in rodents the affinity of α3 isoform of Na+,K+-ATPase for CTSs is three orders of magnitude higher than for the α1 isoform. Thus, in transfected NIH 3T3 fibroblasts, the apparent affinity constant of rat α3-, α2-, and α1-Na+,K+-ATPase for ouabain calculated from [3H]-ouabain binding and ATPase assay yielded values of ~2, 115, and 48,000 nM, respectively [29]. In BALB/c 3T3 cells transfected with rat α3 and α1-Na+,K+-ATPase, Ki values for ouabain estimated by inhibition of 86Rb influx were 8 x 10−8 and 4.5 x 10−5 M, respectively [30]. These differences were confirmed by comparative analysis of the dose-dependent action of ouabain on Na+,K+-ATPase activity in adult and newborn rat brain [31] and in cultured and separated neuronal cells from rat cerebellum [32].

In the present study, we used the sharp differences in the affinity of rat α1 and α3-isoforms of Na+,K+-ATPase for CTSs to examine the relative impact of α1 and α3-mediated excitation–transcription coupling by comparing the action of low and high doses of ouabain on the intracellular Na+ and K+ content and gene expression profile in primary culture of rat cerebellar granule cells.

Materials and methods

Chemicals

Ouabain octahydrate (Cat #O3125) was purchased from Sigma Aldrich (USA) and was diluted to 10 mM in ultra-pure water and further used for cell treatment. Trypsin-EDTA, fetal bovine serum, penicillin-streptomycin, Hank’s Balanced Salt Solution, and trypan blue were purchased from Paneco (Russia). Neurobasal Medium, Supplement B-27, and 0.5 mM GlutaMax were purchased from Gibco (USA). MMLV RT Kit and TaqMan qPCR mix-HS were purchased from Evrogen (Russia). Primers were purchased from DNA-synthesis (Russia). GeneChip Rat Gene 2.0 ST Array; GeneChip WT PLUS Reagent Kit; GeneChipH WT Terminal Labeling Kit; and GeneChip Hybridization, Wash, and Stain Kit were purchased from Affymetrix (USA). Poly-L-ornithine, RIPA buffer, MgCl2, trichloroacetic acid, KCl, reagents for SDS-PAGE, cocktails of protease and phosphatase inhibitors, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide were purchased from Sigma Aldrich. DC Protein Assay Kit was purchased from Bio-Rad (USA). Antibodies phospho-CREB (Ser133) (87G3) Rabbit mAb; β-Actin (D6A8) Rabbit mAb; Bcl-2 Rabbit pAb; Bax Rabbit pAb; and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology (USA). SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Femto Chemiluminescent Substrate, DNase I, and PureLink RNA Mini Kit, SYTO13 were purchased from Thermo Fisher Scientific (USA).

Primary culture of cerebellar granule cells

Commercially available Wistar rats were purchased from Pushchino Animal Incubator (Russia) and kept under standard conditions with natural light regime and freely available water and food. Decapitation was used for euthanasia and all efforts were made to minimize suffering. The animal experiments were approved by the Bioethics Committee of the Faculty of Biology, Lomonosov Moscow State University (#82-O). Cerebellum was isolated from 7- to 8-day-old rat pups under sterile conditions as described previously [33]. Cerebellum cells were dissociated with 0.25% Trypsin-EDTA (Paneco) for 15 min at 37°C. The trypsin was inactivated by incubation with 10% fetal bovine serum (Paneco). Cells were cultivated in Neurobasal-A Medium (Gibco) containing 2% Supplement B-27 (Gibco), 0.5 mM GlutaMax (Gibco), 100 U/ml penicillin-streptomycin (Paneco), and 20 mM KCl plated at density 1.2·105 cells/cm2 onto 6-well plates pretreated with poly-L-ornithine (Sigma). The cells were maintained at 37°C, 90% humidity, and 5% CO2 for 7 or 8 days, at which time they had reached an advanced degree of morphological differentiation [34]. Cytosine arabinoside was added at final concentration 10 μM after 24 h in culture for inhibition of astrocyte proliferation.

Cell death assay

Cells were seeded onto 96-well plates at density 4·104 cells/cm2. After 3 h of ouabain treatment, cells were washed twice in Hank’s Balanced Salt Solution (Paneco). Dead cells were stained with 0.5% trypan blue (Paneco) for 5 min, the live cells were stained for 2 min with 2 μm DNA stain SYTO13 and then cells were washed in Hank’s Balanced Salt Solution. For each experimental point images of fields of view at 200x magnification were obtained in the bright field and upon excitation of fluorescence light with a wavelength of 488 nm (emission 509 nm) for SYTO 13 using Eclipse TS100 fluorescent microscope system (Nikon, Japan). The number of living neurons in culture was assessed as total number of neurons whose nuclei were stained by SYTO 13 but cells were not has been stained with trypan blue.

MTT assay

Cells were seeded onto 96-well plates at density 4·104 cells/cm2. After 3 h of ouabain treatment, the medium was aspirated and replaced by fresh 0.5 mg/ml MTT solution. After 3 h, the MTT solution was removed and the resulting formazan crystals were solubilized by adding 100 μl dimethyl sulfoxide to each well. After 10 min of vigorous vortexing, the absorbance in each well was read using a Synergy H4 microplate reader (Biotek, USA) at 570 nm and 660 nm. Absorbance values at 660 nm were subtracted from absorbance values at 570 nm to correct for nonspecific background.

Quantification of intracellular Na+ and K+ content by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Cells were seeded onto 6-well plates at density 1.2·105 cells/cm2. After 3 h of ouabain treatment, the medium was discarded and the cells were washed twice with cold 0.1 M MgCl2 and lysed in 200 μl of trichloroacetic acid. The lysates were transferred to acid-washed and metal-free 0.5-ml clear microtubes and incubated for 12 h at 4°C. After the incubation, the samples were brought to 500 μl with doubly deionized water. The samples were assayed on a Varian 820-MS (USA). Concentrations of the selected elements (mg/liter) were measured by an external calibration method. The data were analyzed using ICP-MS Expert version 2.1 b-107 (USA). Na+ and K+ contents are expressed as part per million (ppm) per sample.

RNA isolation, reverse transcription, and qPCR

Total RNA was extracted from cells grown in 6-well plates using a PureLink RNA Mini Kit (Ambion) followed by DNase I treatment (Thermo Fisher Scientific) according to the manufacturers’ protocols. One microgram of each RNA sample was reverse transcribed using random decamer primers with an MMLV RT Kit (Evrogen) following the manufacturer’s instructions. The obtained cDNA was used for qRT-PCR, the reaction being carried out with a CXF96 Real-Time PCR System (Bio-Rad Laboratories, USA) and TaqMan qPCR mix-HS (Evrogen). The primers sequences for c-Jun, c-Fos, Bcl-2, Bax, and GAPDH were: c-fos-forward (5’-GACAGCCTTTCCTACTACCAT-3’), c-fos-reverse (5’-GCTGGTGGAGATGGCTGTCA-3’), c-fos (5’-FAM-CTGTCAACACACAGGACTTTTGCGC-BHQ1-3’); c-jun-forward (5’-AAGCTCACAAGTCCCGGCAC-3’), c-jun-reverse (5’-CCTGTGCGAGCTGGTATGAG-3’), c-jun (5’-FAM-GTTCGCTCCGGGCCACTTGTTCC-BHQ1-3’); bcl-2-forward (5’-CACGGTGGTGGAGGAACTCT-3’), bcl-2-reverse (5’-CACATGACCCCACCGAACTC-3’), bcl-2 (5’-FAM -CCACAATCCTCCCCCAGTTCACCC- BHQ1-3’); bax-forward (5’-GGAGACACCTGAGCTGACCT-3’), bax-reverse (5’-ATCGCCAATTCGCCTGAGACA-3’), bax (5’-FAM-CTTCTTGGTGGATGCGTCCTGGGG- BHQ1-3’); GAPDH-forward (5’- ACCCACGGCAAGTTCAACGG-3’), GAPDH-reverse (5’-CCCTTCAGGTGAGCCCCAG-3’), GAPDH (5’- FAM-CGGGATCTCGCTCCTGGAAGATG-BHQ1-3’). The expression of GAPDH mRNA as an internal control was used to normalize and compare the expression value of each gene of interest using the 2–ΔΔCt method. The p-values were calculated using one-way ANOVA with repeated measures and Tukey’s multiple comparison test of all data to the control. Significance was accepted at p<0.05.

Microarray analysis

RNA samples that had more than 7.0 RNA integrity number (RIN) and no detectable genomic DNA contamination were used for the subsequent gene microarray analyses. RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, USA). RNA samples were hybridized to a GeneChip Rat Gene 2.0 ST Array gene expression array (Affymetrix). This array detects total RefSeq 28407 transcripts. Five hundred nanograms of total RNA for each sample was processed with GeneChip WT PLUS Reagent Kit (Affymetrix). This kit uses a reverse transcription priming method that specifically primes non-ribosomal RNAs, including both poly(A) and non-poly(A) mRNAs, and generates amplified and biotinylated sense-stranded DNA as the final product. The single-stranded cDNA (5.5 mg) was fragmented and labeled using an Affymetrix GeneChipH WT Terminal Labeling Kit (Affymetrix), and 3.5 mg of the resulting cDNA was hybridized on the chip using a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Scans of microarrays were converted into CEL files by the scanner software and then jointly preprocessed in the Affymetrix Expression Console (build 1.4.1.46). The expression levels were statistically analyzed using the Affymetrix Transcriptome Analysis Console (version 3.0). The data were initially normalized by the Robust Multichip Average (RMA)-Sketch algorithm, which uses background adjustment, quantile normalization, and summarization. Then, the normalized data were analyzed by principal component analysis (PCA) [35] to identify patterns in the dataset and to highlight similarities and differences among the samples. Major sources of variability identified within the dataset by PCA were used as grouping variabilities for analysis of variance (ANOVA) with n = 3 for each group of samples. The ensuing data were filtered to identify transcripts with statistically significant variation of expression among the groups that are modulated by at least 20%, with multiple testing correction by the false discovery rate (FDR). For interpreting genome-wide expression profiles, Gene Set Enrichment Analysis (GSEA) was applied.

SDS-PAGE and Western blotting

Cells were seeded onto 6-well plates at density 1.2·105 cells/cm2. After 3 h of ouabain treatment, the medium was discarded, and the cells were washed with cold Hank’s Balanced Salt Solution and lysed in RIPA buffer (Sigma) containing cocktails of protease and phosphatase inhibitors (Sigma). The total protein concentration was measured using a DC Protein Assay Kit (Bio-Rad) and 15 μg of protein from cell lysates per sample was separated by 15% SDS-PAGE and transferred to PVDF membrane. Biotinylated proteins served as molecular weight markers (Cell Signaling Technologies, Cat#7727). The membranes were blocked and incubated overnight with primary and appropriate secondary HRP-conjugated antibodies: phospho-CREB (Ser133) (87G3) Rabbit mAb (Cell Signaling Technologies, Cat#9198) at dilution 1:1000; β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology, Cat#8457) at dilution 1:1000; Bcl-2 Rabbit pAb (Cell Signaling Technology, Cat#2876) at dilution 1:1000; Bax Rabbit pAb (Cell Signaling Technology, Cat#2772) at dilution 1:1000; anti-rabbit IgG-HRP (Cell Signaling Technology, Cat#7074) at dilution 1:3000; and anti-biotin, HRP-linked Antibody (Cell Signaling Technology, Cat#7075) at dilution 1:3000. The desired protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate and SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s protocol. Chemiluminescence was detected using the ChemiDoc XRS+ System (Bio-Rad, USA) and intensity counted using Image Lab 3.0 software (Bio-Rad, USA). In each experiment the control value was taken for 100%. β-Actin was used as loading control to ensure that the total amount of protein did not change.

Statistics

The data were analyzed using GraphPad Prism4 Software and by applying one-way ANOVA with repeated measures and Dunnett’s comparison of all data to the control. Significance was accepted at p<0.05.

Results and discussion

Previously, it has been shown that long-term (24 h) exposure to 3 mM ouabain did not affect survival of rat astrocytes, vascular smooth muscle, and endothelial cells, in contrast with the cytotoxic action of ouabain on the human cells. Importantly, transfection of human endothelial cells with rat α1-Na+,K+-ATPase protected them from the cytotoxic action of high doses of ouabain (3–3000 μM) [36]. Consistent with a previous report [37], we observed that 3 h exposure to ouabain did not affect survival of rat cerebellum granule cells (Fig 1A). Thus, the number of trypan blue-stained cells was less than 3–5% in control conditions as well as in the presence of 100 nM or 1 mM ouabain. We also did not observe any significant action of ouabain on the number of neurons stained with SYTO13 (Fig 1B). In additional experiments, we estimated cell viability by MTT assay. We found that 3 h exposure of rat cerebellum granule cells to 1 mM ouabain decreased MTT reduction by 25% (Fig 1C). It should be noted, however, that exposure of rat aortic endothelial cells and primary astrocytes to 5 mM ouabain led to rapid decline of MTT reduction via attenuation of intracellular accumulation of MTT, without any impact on cell survival estimated by chromatin cleavage, caspase-3, and attachment assays [38]. Keeping these data in mind, we quantified the transcription and expression level of pro-apoptotic Bax and anti-apoptotic Bcl-2 by qPCR and Western Blotting, respectively (Fig 2). Bcl-2 family proteins are related to the formation of channels in mitochondrial membranes and regulate cytochrome c release. The released cytochrome c from mitochondria may activate the intrinsic apoptotic pathway via apoptosome formation and caspase-9 activation and thus drive cells to apoptosis. We found that exposure to 1 mM ouabain elevated Bcl-2 transcription by 50% (Fig 2B) without any impact on Bcl-2 protein content (Fig 2D). Neither Bax mRNA (Fig 2A) nor Bax protein (Fig 2E) content was affected by low and high doses of ouabain. Viewed collectively these results show that similar to other rodent cells [37] exposure to ouabain does not affected the survival of rat cerebellum granule cells.

Fig 1

Estimation of viability of rat cerebellum granule cells by trypan blue staining and SYTO13 (A,B) and MTT reduction assay (C).

A) Representative micrographs of control cells and cells exposed to 100 nM and 1 mM ouabain for 3 h. Bars = 100 μm. B) Percentage of SYTO 13-stained control cells and cells exposed to 100 nM and 1 mM ouabain for 3 h. C) MTT reduction assay of control cells and cells exposed to 100 nM and 1 mM ouabain for 3 h. Means ± SD for 4 (B) and 8 (C) independent experiments are shown. *—p<0.05.

Fig 2

Effect of ouabain on content Bcl-2 and Bax mRNAs (A,B) and protein (C-E) in rat cerebellum granule cells.

Cerebellum granule cells were incubated in the absence or presence 100 nM and 1 mM ouabain for 3 h. Bcl-2/GAPDH (A) and Bax/GAPDH (B) ratios in the absence of ouabain were taken as 100%. Representative Western blot (C) and Bcl-2/Actin B (D) and Bax/Actin B (E) ratio. Bcl-2/Actin B and Bax/Actin B ratios in the absence of ouabain were taken as 100%. Means ± SD for 4 independent experiments are shown. *—p<0.05.

Table 1 shows that 3 h inhibition of both α1- and α3-Na+,K+-ATPase in rat cerebellum granule cells by 1 mM ouabain increased intracellular Na+ content 8-fold and decreased [K+]i by 6-fold, whereas selective inhibition of CTS-sensitive α3-Na+,K+-ATPase by 100 nM ouabain had no any significant impact on these parameters. These results suggest that under the baseline conditions, α3-Na+,K+-ATPase has negligible impact on intracellular Na+ and K+ content. This conclusion is consistent with recent data showing very modest increment of [Na+]i in SBFI-loaded cultured rat hippocampal and striatal neurons treated with 1000 nM ouabain, which is in contrast with 7-fold elevation of [Na+]i evoked by 1 mM ouabain [28]. In rat cerebellum granule cells, the ouabain-resistant α1 isoform accounts for about 70%, and the ouabain-sensitive α3 isoform accounts for about 30% of total Na+, K+-ATPase activity in primary rat cerebellar neurons [23]. Thus, it might be proposed that inhibition of the α3 subunit results in local perturbation of Na+ and K+ concentrations, but increased activity of the α1 subunit can compensate for these changes.

Table 1

Effect of ouabain on intracellular Na+ and K+ content in rat cerebellum granule cells.

	[Na+]i content, ppm	[K+]i content, ppm
Control cells	1.15 ± 0.39	11.5 ± 2.93
Ouabain, 100 nM	1.05 ± 0.24	11.2 ± 2.77
Ouabain, 1 mM	9.92 ± 6.49	1.97 ± 0.60

The cells were incubated in the absence or presence of 100 nM or 1 mM ouabain for 3 h. Intracellular Na+ and K+ content in the absence of ouabain was taken as 100%. Mean ± SD obtained in 3 independent experiments.

Exposure of rat cerebellum granule cells to 100 nM or 1 mM ouabain for 3 h resulted in appearance of 144 and 994 differentially expressed transcripts, with the maximal degree of activation and inhibition of 3.22 versus 18.82 and 2.91 versus 2.14, respectively (Table 2) (for complete lists of transcripts whose expression was affected by 100 nM or 1 mM ouabain, see S1 and S2 Tables). Assuming that transcriptomic changes in the presence of 100 nM or 1 mM ouabain are independent events, the number of identified common transcripts (NCOM) might be less than NCOM = (N100 × N1) × NTOT–1, where N100 and N1 are numbers of differential expressed transcripts triggered by 100 nM and 1 mM ouabain (144 and 994, respectively), and NTOT = 28407 is the total transcript number covered by the microarray assay. The calculated number NCOM = 5.04 was higher than the number of common differentially expressed transcripts identified in our study (n = 2; Table 3). This analysis strongly suggests that signaling pathways underlining transcriptomic changes triggered by low and high doses of ouabain are different. This conclusion is supported by data considered below.

Table 2

Number of differentially expressed transcripts in ouabain-treated rat cerebellum granule cells.

	Ouabain, 100 nM	Ouabain, 1 mM
Upregulated
Number of transcripts	73	403
Maximal fold of activation	3.22	18.82
Downregulated
Number of transcripts	71	591
Maximal fold of inhibition	2.14	2.91

Table 3

Genes whose expression was affected by both 100 nM and 1 mM ouabain.

Gene Symbol, Title	Fold of inhibition by 100 nM ouabain / p-value	Fold of inhibition by 1 mM ouabain / p-value
Oxsm, 3-oxoacyl-ACP synthase	–1.34 / 0.030341	–1.66 / 0.031923
OC685989, hypothetical protein	–1.37 / 0.036192	–1.48 / 0.039919

Transcripts whose expression was altered by more than 1.30-fold with p<0.05 were subject to analysis. All experiments were repeated 3 times.

For detailed assessment of effects of low and high doses of ouabain, we performed Affymetrix whole-transcriptome-gene-expression analysis with subsequent functional annotation of the classes of genes that are enriched among the genes differentially regulated in ouabain-treated cells using Gene Set Enrichment Analysis (GSEA). In 1 mM ouabain-treated cells, GSEA revealed at false discovery rate FDR<1%: 269 upregulated (S3 Table) and 103 downregulated (S4 Table) gene sets belonging to the sub-ontology biological process; 32 upregulated (S5 Table) and 16 downregulated (S6 Table) gene sets belonging to sub-ontology molecular function; 1 upregulated (S7 Table) and 8 downregulated (S8 Table) gene sets belonging to the sub-ontology cellular component. We summarized the annotations by removing redundant GO terms and visualized the remaining terms in semantic similarity-based graphs using REViGO [39] with the SimRel measure [40]. We present these graphs in Supporting Information (S1–S6 Figs).

In 100 nM ouabain-treated cells, GSEA revealed almost no significant processes at FDR<1%. We used NES cut-off ± 1.35 to represent the data. In 100 nM ouabain-treated cells, GSEA revealed at FDR<25%: 293 upregulated (S9 Table) and 114 downregulated (S10 Table) gene sets belonging to the sub-ontology biological process; 30 upregulated (S11 Table) and 48 downregulated (S12 Table) gene sets belonging to sub-ontology molecular function; 8 upregulated (S13 Table) and 27 downregulated (S14 Table) gene sets belonging to the sub-ontology cellular component. We represent these graphs in Supporting Information (S7–S12 Figs).

Keeping in mind protein multifunctionality, we limited this analysis to ten major functional categories (Fig 3, Tables 4–7). Fig 3A shows that the genes whose expression was affected by 100 nM ouabain is abundant for olfactory receptors. Olfactory receptors belong to a large gene superfamily comprising approximately 900 genes in the rat genome [41]. Importantly, the relative number of olfactory receptors among differentially expressed genes identified in neurons treated with low doses of ouabain (24%) was 8-fold higher than in the total rat genome. The function of these proteins as seven-transmembrane-type G-protein-coupled receptors is well defined in olfactory epithelium cells. In these highly specialized cells, the binding of pheromones by olfactory receptors leads to activation of adenylate cyclase, elevation of intracellular cAMP, opening of cAMP-gated, Ca2+-permeable channels [42, 43], which in turn, triggers diverse social responses [44]. During the last two decades, expression of olfactory receptors has also been demonstrated in skeletal muscle cells and other non-chemosensory tissues [45]. Recently, we reported that in cultured C2C12 myotubes, both ouabain and electrical pulse stimulation affect expression of olfactory receptors via Ca2+-mediated signaling [46]. The functional significance of altered expression of olfactory receptors in neurons triggered by low doses of ouabain remains unknown.

Fig 3

Distribution of ouabain-sensitive genes detected in cerebellum granule cells and listed in Tables 4–7 among major functional categories.

Cells were incubated in the presence of 100 nM (A) or 1 mM ouabain (B) for 3 h. Functional categories: CPD—cell proliferation and death; IR—immune and inflammatory responses; miRNA—microRNAs; MTR—membrane transporters; O—others; OLFR—olfactory receptors; PSHD—protein, synthesis, holding, and degradation; RP—ribosomal proteins; S—signaling; TR—transcription regulation; UN—uncharacterized.

Table 4

Genes whose expression was increased by 100 nM ouabain by more than 1.3-fold.

Gene Symbol, Title	Functional category	Fold of activation / p-value
Olr551, olfactory receptor 551	OLFR	1.87 / 0.018489
SPC24, NDC80 kinetochore complex component	PSHD	1.55 / 0.04222
Olr1334, olfactory receptor 1334	OLFR	1.5 / 0.037193
RGD1310495, similar to KIAA1919 protein	MTR	1.47 / 0.044616
Olr397, olfactory receptor 397	OLFR	1.47 / 0.014367
LOC102548248, calphotin-like	UN	1.47 / 0.032125
Csdc2, cold shock domain containing C2, RNA binding	TR	1.45 / 0.033715
Olr1394, olfactory receptor 1394	OLFR	1.44 / 0.032407
LOC102556805, rho GTPase-activating protein 20-like	S	1.43 / 0.023919
Drgx, dorsal root ganglia homeobox	TR	1.41 / 0.044668
Akap3, A kinase (PRKA) anchor protein 3	PSHD	1.39 / 0.026649
Map2k4, mitogen activated protein kinase kinase 4	S	1.38 / 0.01881
LOC498601, similar to cyclin B2	UN	1.38 / 0.03072
Snrpf, small nuclear ribonucleoprotein polypeptide F	TR	1.38 / 0.003448
Mageb16, melanoma antigen family B, 16	IR	1.37 / 0.006286
Pnoc, prepronociceptin	S	1.37 / 0.016375
LOC100361079, ribosomal protein L36-like	RP	1.36 / 0.007825
Emd, emerin	PSHD	1.35 / 0.034983
Tcp1-ps1, t-complex protein 1, pseudogene 1	PSHD	1.35 / 0.00873
Rpl22, ribosomal protein L22	RP	1.35 / 0.018718
Akr1c12, aldo-keto reductase family 1, member C12	O	1.35 / 0.031111
Rpl36, ribosomal protein L36	TR	1.34 / 0.043525
Fam174a, family with sequence similarity 174, member A	O	1.33 / 0.028861
Olr352, olfactory receptor 352	OLFR	1.33 / 0.021153
Ctla2a, cytotoxic T lymphocyte-associated protein 2 alpha	PSHD	1.33 / 0.020505
MGC114246, similar to cathepsin R	PSHD	1.33 / 0.045924
LOC102551267, paired box protein Pax-7-like	TR	1.32 / 0.001153
LOC296235, similar to Cystatin S precursor (LM protein)	PSHD	1.32 / 0.010687
Rpl34-ps1, ribosomal protein L34, pseudogene 1	RP	1.32 / 0.049551
Atmin, ATM interactor	TR	1.32 / 0.044838
RGD1559972, similar to ribosomal protein L27a	RP	1.32 / 0.034465
Acot13, acyl-CoA thioesterase 13	O	1.32 / 0.047266
Pcp4, Purkinje cell protein 4	O	1.31 / 0.024075

The cells were incubated in the absence or presence of 100 nM ouabain for 3 h. Functional categories: CPD—cell proliferation and death; IR—immune and inflammatory responses; miRNA—microRNAs; MTR—membrane transporters; O—others; OLFR—olfactory receptors; PSHD—protein, synthesis, holding and degradation; RP—ribosomal proteins; S—signaling; TR—transcription regulation; UN—uncharacterized. All experiments are repeated 3 times.

Table 7

Genes whose expression was decreased by 1 mM ouabain by more than 2-fold.

Gene Symbol, Title	Functional Category	Fold of inhibition / p-value
Downregulated
Otub2, OTU deubiquitinase, ubiquitin aldehyde binding 2	O	–2.01 / 0.049957
Wscd1, WSC domain containing 1	O	–2.01 / 0.014985
Prkag2, protein kinase, AMP–activated, gamma 2 subunit	S	–2.01 / 0.039038
Mki67, marker of proliferation Ki-67	UN	–2.03 / 0.036377
Mavs, mitochondrial antiviral signaling protein	IR	–2.1 / 0.031824
Eya2, EYA transcriptional coactivator and phosphatase 2	TR	–2.13 / 0.035164
Zfp703, zinc finger protein 703	TR	–2.16 / 0.047628
RGD1308147, similar to expressed sequence AW209491	UN	–2.19 / 0.000092
Mir181b1, microRNA 181b-1	miRNA	–2.21 / 0.038541
Ptpn14, protein tyrosine phosphatase, non-receptor type 14	S	–2.21 / 0.03322
Ddit4l, DNA-damage-inducible transcript 4-like	S	–2.23 / 0.029361
Mir421, microRNA 421	miRNA	–2.24 / 0.019258
Nudt13, nudix (nucleoside diphosphate linked moiety X)-type	O	–2.25 / 0.020326
Mir410, microRNA 410	miRNA	–2.27 / 0.000698
Rabif (RGD1563962), RAB interacting factor	S	–2.28 / 0.03624
Pth1r, parathyroid hormone 1 receptor	S	–2.29 / 0.000959
Gdpgp1, GDP-D-glucose phosphorylase 1	O	–2.31 / 0.010957
Vof16, ischemia related factor vof-16	UN	–2.35 / 0.006096
LOC100359748, zinc finger CCCH type, antiviral 1	O	–2.36 / 0.004982
Mir544, microRNA 544	miRNA	–2.36 / 0.010114
Grtp1, growth hormone regulated TBC protein 1	S	–2.41 / 0.036684
Iqub, IQ motif and ubiquitin domain containing	S	–2.42 / 0.027833
Rcor2 (Rcor2l1), REST corepressor 2	TR	–2.44 / 0.035033
Rxra, retinoid X receptor alpha	CPD	–2.48 / 0.028688
Adgra1, adhesion G protein-coupled receptor A1	S	–2.48 / 0.000943
Mir434, microRNA 434	miRNA	–2.51 / 0.004484
Krcc1, lysine-rich coiled-coil 1	TR	–2.57 / 0.012588
Atoh8, atonal bHLH transcription factor 8	TR	–2.68 / 0.03377

The cells were incubated in the absence or presence of 1 mM ouabain for 3 h. For functional categories, see Table 4. All experiments were repeated 3 times.

Unlike for low dose, for 1 mM ouabain the list of differentially expressed genes was abundant in intermediates of intracellular signaling, transcription regulators, and proteins involved in inflammatory and immune responses (26%, 25%, and 11%, respectively) (Tables 6 and 7; Fig 3B). Among transcription regulators whose expression was increased more than 3-fold, we found Npas4, Zfp36, Klf6, Fosb, Junb, Klf4, Nfkbia, and Atf3 (Table 6). We also observed a modest elevation of mRNAs encoding several other immediate-early-response genes (IERGs) including cAMP responsive element transcription regulator (Crem) and CREB/ATF bZIP transcription factor (Crebzf) (S2 Table).

Table 6

Genes whose expression was increased by 1 mM ouabain by more than 2-fold.

Gene Symbol, Title	Functional Category	Fold of activation / p-value
Il6, interleukin 6	IR	18.82 / 0.017424
Ptgs2, prostaglandin-endoperoxide synthase 2	IR	16.59 / 0.00836
Tfpi2, tissue factor pathway inhibitor 2	PSHD	15.82 / 0.035903
Npas4, neuronal PAS domain protein 4	TR	12.97 / 0.013906
Gem, GTP binding protein overexpressed in skeletal muscle	S	8.45 / 0.014514
Cyr61, cysteine-rich, angiogenic inducer, 61	IR	8.09 / 0.005394
Zfp36, zinc finger protein 36	TR	7.56 / 0.032644
Ccl7, chemokine (C-C motif) ligand 7	IR	6.88 / 0.002458
Has2, hyaluronan synthase 2	O	6.84 / 0.001877
Cxcl1, chemokine (C-X-C motif) ligand 1	IR	5.62 / 0.014029
Klf6, Kruppel-like factor 6	TR	5.52 / 0.025668
Adamts1, ADAM metallopeptidase with thrombospondin motif	PSHD	5.08 / 0.005289
Ptx3, pentraxin 3, long	IR	5.02 / 0.00991
Inhba, inhibin beta-A	CPD	4.94 / 0.018744
Bhlhe40, basic helix-loop-helix family, member e40	TR	4.34 / 0.034122
Trib1, tribbles pseudokinase 1	S	4.33 / 0.00474
Coq10b, coenzyme Q10B	PSHD	4.32 / 0.001009
Junb, jun B proto-oncogene	TR	4.22 / 0.00198
Fosb, FBJ osteosarcoma oncogene B	TR	4.21 / 0.021086
Procr, protein C receptor, endothelial	S	4.12 / 0.030968
Rd3l, retinal degeneration 3-like	UN	3.94 / 0.031525
Per1, period circadian clock 1	CPD	3.9 / 0.024427
Cxcl6, chemokine (C-X-C motif) ligand 6	IR	3.86 / 0.027669
Emp1, epithelial membrane protein 1	O	3.68 / 0.033698
Ccnl1, cyclin L1	CPD	3.56 / 0.008671
Dusp6, dual specificity phosphatase 6	S	3.55 / 0.018941
Klf4, Kruppel-like factor 4 (gut)	TR	3.49 / 0.020354
Spry4, sprouty RTK signaling antagonist	S	3.48 / 0.004545
Vgf, VGF nerve growth factor inducible	CPD	3.32 / 0.023502
Nfkbia, nuclear factor of kappa light polypeptide gene enhancer	TR	3.14 / 0.001767
Atf3, activating transcription factor 3	TR	3.02 / 0.021591
Rnf122, ring finger protein 122	PSHD	2.94 / 0.028845
Slc25a25, mitochondrial phosphate carrier	MTR	2.92 / 0.016024
Rgs16, regulator of G-protein signaling 16	S	2.89 / 0.027214
Pnrc1, proline-rich nuclear receptor coactivator 1	TR	2.87 / 0.005126
Mstn, myostatin	IR	2.87 / 0.049177
Spry1, sprouty RTK signaling antagonist 1	S	2.84 / 0.031365
Rnd3, Rho family GTPase 3	S	2.83 / 0.010714
Jun, jun proto-oncogene	TR	2.78 / 0.001458
Pfkfb3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3	S	2.77 / 0.018456
Rel, v-rel avian reticuloendotheliosis viral oncogene homolog	TR	2.67 / 0.012933
Plk3, polo-like kinase 3	S	2.63 / 0.002178
Pmaip1, phorbol-12-myristate-13-acetate-induced protein 1	CPD	2.61 / 0.026542
Icam1, intercellular adhesion molecule 1	IR	2.6 / 0.035928
Gch1, GTP cyclohydrolase 1	S	2.53 / 0.002142
Lysmd3, LysM, putative peptidoglycan-binding	UN	2.48 / 0.034801
Tmem2, transmembrane protein 2	O	2.47 / 0.035654
LOC102552920, armadillo repeat-containing X-linked protein	UN	2.45 / 0.021451
Plau, plasminogen activator, urokinase	PSHD	2.43 / 0.014206
Arid5b, AT rich interactive domain 5B (Mrf1-like)	TR	2.35 / 0.02648
Xirp1, xin actin-binding repeat containing 1	O	2.35 / 0.039713
Fgf9, fibroblast growth factor 9	CPD	2.33 / 0.032372
Ldlr, low density lipoprotein receptor	S	2.31 / 0.000417
Rcan1, regulator of calcineurin 1	S	2.27 / 0.009104
Lysmd3, LysM, putative peptidoglycan-binding	UN	2.21 / 0.017634
Rgs2, regulator of G-protein signaling 2	S	2.19 / 0.012669
Il1ra, interleukin 1 receptor accessory protein	IR	2.15 / 0.029089
Slc39a14, solute carrier family 39 (zinc transporter), member 14	MTR	2.14 / 0.012704
Thbs2, thrombospondin 2	IR	2.13 / 0.020824
Arl5b, ADP-ribosylation factor-like 5B	S	2.12 / 0.041265
Gcnt4, glucosaminyl (N-acetyl) transferase 4, core 2	O	2.12 / 0.006454
Il1r1, interleukin 1 receptor, type I	IR	2.11 / 0.041316
Zfp711, zinc finger protein 711	TR	2.08 / 0.025828
Clcn5, chloride channel, voltage-sensitive 5	MTR	2.06 / 0.020143
Arrdc3, arrestin domain containing 3	S	2.05 / 0.012561
Timp1, TIMP metallopeptidase inhibitor 1	PSHD	2.05 / 0.01694
Gucy1a2, guanylate cyclase 1, soluble, alpha 2	S	2.04 / 0.02076
Ahr, aryl hydrocarbon receptor	TR	2.04 / 0.020671
Alkbh1, alkB homolog 1, histone H2A dioxygenase	TR	2.02 / 0.046413
Mxd, max dimerization protein 1	TR	2.01 / 0.04203
LOC498465, similar to RIKEN cDNA 1700001F09	UN	2.01 / 0.011855

The cells were incubated in the absence or presence of 1 mM ouabain for 3 h. For functional categories, see Table 4. All experiments were repeated 3 times.

The Zfp36 (TTP) gene encodes an RNA-binding protein that regulates the metabolism of mRNAs by targeting them for degradation. TTP interacts with many mRNAs encoding a wide range of growth factors, cytokines, and proto-oncogenes and destabilizes them. It has been shown that TTP downregulation is necessary for proper neuronal differentiation in vitro and might be a novel post-transcriptional repressor of nervous-system-specific genes that participate during brain development [47].

Activating transcription factor 3 (Atf3) belongs to the ATF/CREB transcription factor family involved in cell growth and apoptosis. It has been shown that ATF3 is a downstream target of the JNK/c-Jun pathway and contributes to apoptosis induced by potassium deprivation in rat cerebellum cell culture [48]. Numerous studies have demonstrated that augmented expression of these genes might be involved in learning and memory consolidation via modulation of synaptic plasticity and adult hippocampal neurogenesis [49, 50]. Another upregulated IERG Kruppel-like factor 4 (Klf4) is also involved in cell proliferation, differentiation, and death. Zhu et al demonstrated that glutamatergic stimulation of cultured cortical neurons results in overexpression of KLF4, which regulates cell cycle proteins and sensitizes neurons to NMDA-induced caspase-3 activity [51].

Bdnf encodes a neurotrophin that plays a key role in neuronal development [52, 53]. We observed that 1 mM ouabain evokes 13-fold increase of Npas4, which was found to be associated with Bdnf promoters I and IV (Table 6). It has been shown that Npas4 expression is rapidly activated by excitatory synaptic activity and turns on a program of gene expression that triggers the formation and/or maintenance of inhibitory synapses on excitatory neurons [54].

Table 3 shows that the list of transcripts affected by both 100 nM and 1 mM ouabain is limited to two down-regulated genes (Oxsm and OC685989). Based on the UniProt database, Oxsm protein may be involved in biosynthesis of lipoic acid as well as longer chain fatty acids required for optimal mitochondrial function [55]. The function of hypothetical OC685989 protein remains unknown.

Transcriptomic changes in cerebellum granule cells treated with 1 mM ouabain are probably mediated by inhibition of α1-Na+,K+-ATPase and elevation of the [Na+]i/[K+]i ratio. This conclusion is supported by comparative analysis of dose- and time-dependent action of ouabain and another cardiotonic steroid, marinobufagenin, on intracellular content of Na+ and K+ and gene expression in human endothelial cells abundant with α1-Na+,K+-ATPase [56]. In several types of cells, elevation of the [Na+]i/[K+]i ratio increases [Ca2+]i via activation of an Na+/Ca2+ exchanger [57] and/or voltage-gated Ca2+ channel [58]. Thus, it might be assumed that excitation–transcription coupling triggered by high doses of ouabain is “at least partially” driven by changes in [Ca2+]i and activation of several Ca2+-mediated pathways including phosphorylation of cAMP response-element-binding protein CREB (for reviews, see [59, 60]). Fig 4 shows that 1 h exposure to 1 mM ouabain increased CREB phosphorylation by ~20-fold, thus suggesting an impact of Ca2+- and/or cAMP-mediated signaling.

Fig 4

Effect of ouabain (oua) on CREB phosphorylation in cerebellum granule cells.

A representative Western blot (A) and means ± SD for 4 independent experiments (B) are shown. *–p<0.05 compared to control.

The presence of cAMP-response element (CRE) within promoters of diverse IERGs including c-Fos and c-Jun B is well-documented [61, 62]. Consistent with microarray data (Table 6), we documented a sharp increment of these gene transcripts in cells treated with 1 mM ouabain using the qPCR approach (Fig 5). It should be noted, however, that in vascular smooth muscle and endothelial cells, augmented expression of these and several other IERGs were preserved in the presence of extra- and intracellular Ca2+ chelators [63-65] and selective inhibitors of calmodulin and Ca2+-sensitive protein kinase and phosphatases [58]. Thus, additional experiments should be performed to examine the relative impact of [Ca2+]i-dependent and -independent signaling in transcriptomic changes triggered in rat cerebellum granule cells by α1-Na+,K+-ATPase-mediated elevation of the [Na+]i/[K+]i ratio.

Fig 5

Expression of c-Fos (A) and c-Jun (B) mRNAs in cerebellum granule cells after 3 h of incubation with 100 nM or 1 mM ouabain.

Data obtained in 5 independent experiments are presented as the mean ± SD. Gene expression in the absence of ouabain was taken as 100%.

Considering the physiological implications of the extended [Na+]i,[K+]i-sensitive transcriptome identified in our study, it should be noted that side-by-side with α1-Na+,K+-ATPase inhibition, dissipation of transmembrane gradients of monovalent cations might be triggered by activation of monovalent cation-permeable ionotropic AMPA receptors [66] and voltage-gated Na+ channels [67, 68]. Indeed, using acute tissue slices of mouse cerebellum loaded with fluorescent Na+ indicators, it was shown that in dendrites of Purkinje neurons glutamate transiently increases [Na+]i by 5–10 mM via activation of AMPA receptors [69]. In Purkinje neurons, transient increments of [Na+]i were also evoked by brief (50 Hz/100 ms) electrical pulse stimulation [46]. In dopamine cells of the substantia nigra pars compacta, electrical hyperpolarization induced a rise in [Na+]i from 10 to 25 mM [70]. Because NMDA receptors exhibit low cation selectivity (PNa ~ PK> PCa), they also contribute to elevation of the [Na+]i/[K+]i ratio triggered by synaptic excitation. Consistent with these data, selective activation of NMDA receptors in rat cerebellar granule cells resulted in reversible elevation of [Na+]i from 5 to 60 mM [71]. Using cultured cerebellar neurons, Linden et al. showed that an increase in [Na+]i rather than [Ca2+]i is required for the induction of long-term depression [72].

Modest transcriptomic changes evoked by 100 nM ouabain suggest implication of the α3 isoform as an upstream intermediate of signal transduction. Importantly, at this concentration, ouabain did not affect intracellular content of monovalent cations (Table 1). Recent studies have revealed that CTSs can affect cells independently of suppression of Na+,K+-ATPase-mediated ion fluxes. Thus, at low concentrations ouabain triggered the interaction of the Na+,K+-ATPase α-subunit with the membrane-associated nonreceptor tyrosine kinase Src, activation of Ras/Raf/ERK1,2, phosphatidyl inositol 3-kinase (PI(3)K), PI(3)K-dependent protein kinase B, phospholipase C, and [Ca2+]i, and oscillations and augmented production of reactive oxygen species (for reviews, see [16, 17, 19, 73]). Earlier it was shown that in rat cerebellum granule cells, 100 nM ouabain activates MAP kinase via PKC and PIP(3) kinase, whereas 1 mM ouabain regulates this MAP kinase signaling cascade via an Src kinase-dependent pathway [74]. Unlike 1 mM ouabain, we did not detect any elevation of CREB phosphorylation in the presence of 100 nM ouabain (Fig 4). The role of other intermediates of [Na+]i/[K+]i-independent signaling in transcriptomic changes triggered by low doses of ouabain should be examined in forthcoming studies.

In conclusion, we report here that complete inhibition of Na+,K+-ATPase by 1 mM ouabain results in dissipation of transmembrane Na+ and K+ gradients, whereas selective inhibition of α3- Na+,K+-ATPase by 100 nM ouabain has no impact on the [Na+]i/[K+]i ratio. Among hundreds of differentially expressed transcripts detected in the presence of high and low doses of ouabain, we identified only 2 common genes, thus indicating different mechanisms of excitation–transcription coupling mediated by α1- and α3-Na+,K+-ATPase. Among genes sharply upregulated by 1 mM ouabain, we found Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose augmented expression was demonstrated in neurons. The list of genes affected by 100 nM ouabain is abundant in olfactory receptors, whose function in neuronal cells remains poorly understood. Downstream mediators of [Na+]i/[K+]i-independent excitation–transcription coupling signaling triggered by interaction of ouabain with α3-Na+,K+-ATPase remains unknown.

Summary of upregulated gene sets (GeneOntology—Biological process) in 1mM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Biological process) in 1mM ouabain-treated granular neurons.

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Summary of upregulated gene sets (GeneOntology—Molecular function) in 1mM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Molecular function) in 1mM ouabain-treated granular neurons.

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Summary of upregulated gene sets (GeneOntology—Cellular component) in 1mM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Cellular component) in 1mM ouabain-treated granular neurons.

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Summary of upregulated gene sets (GeneOntology—Biological process) in 100nM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Biological process) in 100nM ouabain-treated granular neurons.

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Summary of upregulated gene sets (GeneOntology—Molecular function) in 100nM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Molecular function) in 100nM ouabain-treated granular neurons.

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Summary of upregulated gene sets (GeneOntology—Cellular component) in 100nM ouabain-treated granular neurons.

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Summary of downregulated gene sets (GeneOntology—Cellular component) in 100nM ouabain-treated granular neurons.

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Transcripts whose expression was change by more than 1.3-fold by 100 nM ouabain.

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Transcripts whose expression was change by more than 1.3-fold by 1 mM ouabain.

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Upregulated gene sets (GeneOntology—Biological process) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Downregulated gene sets (GeneOntology—Biological process) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Upregulated gene sets (GeneOntology—Molecular function) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Downregulated gene sets (GeneOntology—Molecular function) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Upregulated gene sets (GeneOntology—Cellular component) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Downregulated gene sets (GeneOntology—Cellular component) in 1mM ouabain-treated granular neurons significant at FDR < 1%.

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Upregulated gene sets (GeneOntology—Biological process) in 100nM ouabain-treated granular neurons at NES < -1.35.

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Downregulated gene sets (GeneOntology—Biological process) in 100nM ouabain-treated granular neurons at NES > 1.35.

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Upregulated gene sets (GeneOntology—Molecular function) in 100nM ouabain-treated granular neurons at NES < -1.35.

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Downregulated gene sets (GeneOntology—Molecular function) in 100nM ouabain-treated granular neurons at NES > 1.35.

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Upregulated gene sets (GeneOntology—Cellular component) in 100nM ouabain-treated granular neurons at NES < -1.35.

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Downregulated gene sets (GeneOntology—Cellular component) in 100nM ouabain-treated granular neurons at NES > 1.35.

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Table 5

Genes whose expression was decreased by 100 nM ouabain by more than 1.3-fold.

Gene Symbol, Title	Functional category	Fold of inhibition / p-value
Olr1393, olfactory receptor 1393	OLFR	–1.31 / 0.007599
Olr587, olfactory receptor 587	OLFR	–1.31 / 0.035127
Olr1410, olfactory receptor 1410	OLFR	–1.31 / 0.02515
Cox6c–ps1, cytochrome c oxidase subunit VIc, pseudogene	O	–1.31 / 0.016536
Usp49, ubiquitin specific peptidase 49	PSHD	–1.31 / 0.036476
Tmem266, transmembrane protein 266	UN	–1.31 / 0.02683
Gnb1l, guanine nucleotide binding protein (G protein)	S	–1.31 / 0.005584
Sntb1, syntrophin, beta 1	PSHD	–1.32 / 0.027004
LOC102549817, zinc finger protein 124-like	TR	–1.33 / 0.047193
LOC103690541, uncharacterized	UN	–1.33 / 0.011505
RGD1311300, similar to T cell receptor V delta 6	UN	–1.34 / 0.014249
Lrrc72, leucine rich repeat containing 72	UN	–1.34 / 0.00591
Oxsm, 3–oxoacyl-ACP synthase, mitochondrial	O	–1.34 / 0.030341
Olr1695, olfactory receptor 1695	OLFR	–1.35 / 0.034048
Cyyr1, cysteine / tyrosine–rich 1	UN	–1.35 / 0.037929
Olr1374, olfactory receptor 1374	OLFR	–1.36 / 0.031302
Defb25, defensin beta 25	IR	–1.36 / 0.016742
Defb41, defensin beta 41	IR	–1.36 / 0.042335
Spata31e1, SPATA31 subfamily E, member 1	CPD	–1.36 / 0.0433
Ric8b, RIC8 guanine nucleotide exchange factor B	S	–1.37 / 0.042087
LOC689679, similar to Discs large homolog	UN	–1.37 / 0.010808
LOC685989, hypothetical protein LOC685989	UN	–1.37 / 0.036192
Snap25, synaptosome–associated protein 25	MTR	–1.39 / 0.036352
Olr1192, olfactory receptor 1192	OLFR	–1.39 / 0.00279
Zkscan3, zinc finger with KRAB and SCAN domains 3	TR	–1.39 / 0.02762
Olr1875, olfactory receptor 1875	OLFR	–1.40 / 0.039398
Cdkl5, cyclin-dependent kinase-like 5	S	–1.41 / 0.008764
Olr687, olfactory receptor 687	OLFR	–1.42 / 0.015524
Olr639, olfactory receptor 639	OLFR	–1.44 / 0.03603
Olr1065, olfactory receptor 1065	OLFR	–1.44 / 0.033742
RGD1566085, similar to pyridoxal kinase	O	–1.47 / 0.044618
Olr1246, olfactory receptor 1246	OLFR	–1.54 / 0.015191
Slc6a12, solute carrier family 6 neurotransmitter transporter	MTR	–1.56 / 0.015467

The cells were incubated in the absence or presence of 100 nM ouabain for 3 h. For functional categories see Table 4. All experiments are repeated 3 times.