Fig. 4
OCT4 directly bound to BMP2 promoter and decreased its CpG methylation. A, B Agarose gel electrophoresis showed the PCR products of input and OCT4-ChIP; positive control (PC) was performed by amplifying the GAPDH promoter in RNA polymerase II (RPII)-ChIP; normal mouse IgG as a negative control. BMP2-PBS1/2/3/4 = BMP2 promoter binding site 1/2/3/4; SMAD4-PBS1 = SMAD4 promoter binding site 1. C Q-PCR was used to analyze the result of ChIP (n = 3), and the data were expressed as the fold change to the negative control. D–E QRT-PCR and immunoblotting (IB) analysis of BMP2 expression of human renal interstitial fibroblasts (hRIFs) transfected with Len-OCT4 or Len-sh-OCT4 (n = 3). F IHC staining for BMP2 of the normal control (NC) group (granules with hRIFs), the Len-ctrl group (granules with hRIFs transfected with Len-ctrl), and the Len-OCT4 group (granules with hRIFs transfected with Len-OCT4), Len-sh-ctrl group (granules with hRIFs transfected with Len-sh-ctrl) and the Len-sh-OCT4 group (granules with hRIFs transfected with Len-sh-OCT4); n = 6. G A schematic diagram illustrating the pGL3-basic luciferase reporter vector carrying the predicted sequence of BMP2-PBS4 (BMP2-PBS4-wt) or the mutant sequence (BMP2-PBS4-mut). H P-ctrl or p-OCT4 and pGL3-BMP2-PBS4-wt or pGL3-BMP2-PBS4-mut were co-transfected to hRIFs, and the relative luciferase activity (firefly/renilla) was determined (n = 3). I Two predicted CpG islands in BMP2 promoter. J–L Len-sh-OCT4 or Len-sh-ctrl transfected hRIFs were induced with osteogenic medium for 7 days, and bisulfite sequencing PCR (BSP) was used to analyze the CpG methylation in predicted islands (n = 3). Black circle = methylation site; white circle = unmethylation site. M, N HRIFs were co-transfected with either Len-ctrl or len-OCT4 in conjunction with Len-sh-ctrl or Len-sh-BMP2, and IB determined the protein expression of OCT4, OCN, BMP2, and RUNX2 7 days after osteogenic induction (n = 3); O Alizarin Red Staining (ARS) for calcium nodes 14 days after osteogenic induction (n = 3)