Fig. 4

E2F6 recruits KDM5C to the GpC island of the SF3A3 promoter and induces histone demethylation. A T24 cells were transfected with pKLO, shKDM5C, or J82 with pWPI and oeKDM5C vectors, and KDM5C and SF3A3 proteins level were measured using Western blot. B Quantitative analysis of protein expression in A. C T24 and J82 cells were transfected as in A and KDM5C, and SF3A3 mRNA expression was examined by RT-qPCR. D ChIP-PCR with H3K4me2 or IgG detected the CpG isolate of SF3A3 promoter in pWPI or oeSF3A3 vectors transfected-J82 cells. E T24 and J82 were transfected with pWPI or oeKDM5C vectors, and then, bisulfite sequencing PCR (BSP) was used to examine the DNA methylation in the GpC island of the SF3A3 promoter. F J82 cells were transfected with pWPI or oeKDM5C and a coimmunoprecipitation coupled with mass spectrometry (CoIP-MS) was performed with KDM5C antibody. The down table exhibits 7 proteins that increased interaction with KDM5C after overexpression of KDM5C. G CoIP-Western blot detected the interaction between KDM5C and E2F6 after KDM5C overexpression. H T24 and J82 were transfected as in A and then DNA methylation condition was measured using BSP in the GpC island of SF3A3 promoter. All data are expressed as mean ± SEM and come from three independent experiments. *P < 0.05, **P < 0.01 vs. the corresponding controls