Figure 4.

Plasma membrane depolarization, like oncogenic stress, turns off mitotic genes. (a) Heatmap representation showing the expression pattern of 532 differentially expressed genes shared by HEC‐TM cells treated either with KCl during 24 hr (KCl‐24hr) or with 4‐OHT during 96 hr (4‐OHT‐96 hr) compared to untreated HEC‐TM cells (Ctrl), and HEC‐TM transduced with a shRNA against SCNA9 treated with 4‐OHT for 96 hr (4‐OHT‐96 hr shSCN9A). Differentially expressed genes for KCl‐24 hr and 4‐OHT‐96 hr were identified from Agilent microarray data using unpaired t test analysis with Bonferroni multiple testing correction with fold change ≥2.0 and a p value cutoff of <.01. Common regulated genes were selected following a Venn diagram analysis. Pseudocolors indicate differential expression compared to HEC‐TM untreated cells sample (ctrl) (blue indicates downregulated genes and red upregulated transcripts). This dendrogram shows the hierarchical clustering (unsupervised; Pearson centered correlation with centroid linkage rules). (b) Venn diagram comparing the gene sets enriched for downregulated genes in KCl‐24 hr and 4‐OHT‐96 hr conditions according to GSEA (FDR <5%). (c) A gene set enrichment analysis (GSEA) was performed for 1,039 GO gene sets using the log2‐transformed fold change of expression between control and KCl‐24 hr cells as well as between controls and 4‐OHT‐96 hr cells (see Methods). The “Phase M” pathway (from GO) was one of the most significant gene sets that were downregulated in KCl‐24 hr and 4‐OHT‐96 hr compared to control samples, respectively. (d) The enrichment score of the “Phase_M” pathway from Gene Ontology was computed in each sample, using the ssGSEA tool (see Method). The scores were statistically compared between the three replicates of the different experimental conditions: control, KCl‐24 hr, 4‐OHT‐96 hr, and 4‐OHT‐96 hr shSCN9A. (e) Cells were treated with 65 mM KCl for 0, 8, or 24 hr before RNA extraction. RT‐qPCR was then performed on the indicated genes, and their levels of expression were normalized against GAPDH mRNA levels. This experiment was performed three times. Statistical analysis was performed with the Student's t test, ** means p < .01, ***p < .001