Fig. 5.

SOX9 regulates the expression of TNFRSF11A. a The microarray data from VCAP cells transfected with con-siRNA or SOX9-siRNA was used to examine the differentially expressed mRNAs. 1118 differentially expressed genes including 409 up-regulated and 709 down-regulated were identified by the Limma R package and the top 200 differentially expressed mRNAs were selected to draw a heatmap. b The intersection of the common genes between 709 down-regulated gene caused by SOX9 downregulation and 100 SOX9 co-expression genes identified 4 putative substrates of SOX9. c The correlation of the mRNAs of SOX9 and TNFRSF11A in 492 prostate cancer tissues from the TCGA database based on the GEPIA2 website (http://gepia2.cancer-pku.cn). d LNCaP cells were transfected with indicated siRNAs for 36 h, the relative mRNA expression levels of SOX9 and TNFRSF11A were determined by real-time PCR assay. **P < 0.01, ***P < 0.001. e LNCaP cells were transfected with indicated siRNAs for 36 h, and then subjected to western blot with indicated antibodies. f LNCaP cells were transfected with vector control or Flag-SOX9 plasmids for 36 h, the relative mRNA expression levels of SOX9 and TNFRSF11A were determined by real-time PCR assay. ***P < 0.001. g LNCaP cells were transfected with vector control or Flag-SOX9 plasmids for 36 h, and then subjected to western blot with indicated antibodies. h Schematic diagram shows the putative SOX9 response elements from the human TNFRSF11A gene promoter. i The human TNFRSF11A promoter contains two SOX9 response elements. Point mutation was highlighted with black cross. SOX9 was co-transfected with indicated plasmids into LNCaP cells for 36 h. The luciferase activity was then measured. j ChIP assay shows enrichment of SOX9 at the human TNFRSF11A gene promoter in LNCaP cells. The human GAPDH promoter was served as a negative control. ***P < 0.001. The primer pairs for ChIP assay were also demonstrated in h