Fig. 5

MAX recruits PCGF6 and KDM5D to the promoter region and facilitates MAZ hypomethylation. A TCGA data were used to determine MAZ promoter methylation levels in tumor and healthy kidney tissues. B Methprimer was used to analyze the CpG island within MAZ promoter (1400 bp). C DNA methylation of MAZ promoter was examined using bisulfite sequencing PCR (BSP). D Caki-2 cells were transfected with shPCGF6-1# (shPCGF6) or control vector, and then ChIP-PCR was used to explore the CpG isolate of MAZ promoter with IgG or H3K4me3 antibody. E DNA methylation in CpG island of the MAZ promoter was measured by bisulfite sequencing PCR (BSP) in Caki-2 and ACHN with indicated transfection. F Cells overexpressed with PCGF6 were immunoprecipitated with PCGF6 antibody and then analyzed using CoIP-MS. There are 13 proteins listed in the table that interact more strongly with PCGF6 after overexpression of PCGF6. G PCGF6, MAX, and KDM5D interaction were detected by the CoIP-Western blot. H After transfection of Caki-2 and ACHN with oePCGF6 or shMAX or corresponding control vectors, the DNA methylation condition of the CpG island of the MAZ promoter was examined using BSP. I Potential binding sites of MAX within CpG island of MAZ promoter were analyzed using the Ensembl and PROMO 3.0 websites. J With antibodies against PCGF6, KDM5D, and MAX, ChIP-PCR was used to determine the binding site of MAX/PCGF6/KDM5D complex on CpG island. K ACHN cells co-transfected with the indicated vectors were used for the luciferase reporter assays. *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding controls