Figure 2
ESS2 regulated NF-κB/CHD1 target genes. (a) Heatmap data of prostate cancer-related genes by microarray analysis in PC3-shC and PC3-shESS2 cells. (b) RT-qPCR of TMPRSS2 in PC3-shC and PC3-shESS2 cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. (c) Heatmap data of NF-κB/CHD1 target genes by microarray analysis in PC3-shC and PC3-shESS2 cells. (d) Fold changes in gene expression levels (PC3-shESS2/PC3-shC) in (c). (e) RT-qPCR of TNF and IL6 in PC3-shC and PC3-shESS2 cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. (f) Western blotting of p65, CHD1, and β-actin in PC3-shC and PC3-shESS2 cells. Raw data was shown in Supplementary Fig. 8. (g) ChIP-qPCR analysis of TNF at the TSS site (TSS) and 3′-UTR site (3′-UTR) with anti-p65, anti-CHD1, and anti-histoneH3K36 trimethylation antibodies in PC3-shC and PC3-shESS2 cells normalized to the input level. *, p < 0.05, Student’s t-test. (h) Luciferase reporter assays using a p65/p50 expression vectors and/or recombinant TNFα to activate NF-κB on NF-κB response element (NFκRE)-luc reporter vector in HEK293 cells. (i) Luciferase reporter assays using a NFAT expression vector to activate NFAT on NFAT response element (NFAT RE)-luc reporter vector in HEK293 cells. (j) Luciferase reporter assays using 1 μM phorbol 12-myristate 13-acetate (PMA) to activate AP-1 on AP1 response element (AP1 RE)-luc reporter vector in HEK293 cells. (k) ESS2 knockdown by esiRNA decreased the transcriptional activities of NFAT in HEK293 cells. (l) RT-qPCR of ESS2 in PC3-shC and PC3-shESS2 cells treated with/without 1 μM ionomycin for 24 h, normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. (m) Scatter plots showing correlations between ESS2 and NF-κB/CHD1 target gene expression (IER3, LIF, and CSF2) in patients with prostate cancer. For RT-qPCR, ChIP-qPCR and luciferase reporter assays, each experiment was performed at least three times, and the results are presented as means ± standard deviations.
