Figure 4

Early Growth Response 3 regulates Il6 and IL8 in M12 prostate cancer cells. (A) A Venn diagram compares the list of genes that were induced by EGR3 in M12 cells with the list of genes that were highly correlated with EGR3 in clinical human prostate cancer samples. The list of nine genes found at the intersection is shown below the diagram. (B) Confirmation of EGR3-mediated regulation of gene expression. Total RNA was extracted from stable cell lines control (shSCR-M12) and shEGR3 (shEGR3-cl2 and shEGR3-cl3). The mRNA expression was analysed by quantitative RT–PCR using specific primers. The housekeeping gene GAPDH was used as reference. Expression values obtained for each shEGR3 clone were compared with shSCR control using the 2^−ΔΔCT method. (C) Chromatin immunoprecipitation of EGR3 target genes IL6 and IL8. Cells fixed with paraformaldehyde and a specific EGR3 antibody was used to capture protein–DNA complexes. Nonimmune IgG was used as negative control. Promoter regions were amplified by real-time qPCR and analysed by electrophoresis on agarose gels.