Figure 3.

P300/c-Jun induces an upregulation of COL6A1 by modulating H3K27 acetylation. A. The expression of COL6A1 was detected by western blot and qRT-PCR upon treatment of different dose of NaBu in OS cell lines, U2OS and Saos-2. B. ChIP assay demonstrated that H3K27 acetylation occurred in the promoter of COL6A1 in OS cell lines using two primers upon treatment of NaBu. C. ChIP assay demonstrated that H3K27 acetylation occurred in the promoter of COL6A1 in OS cell lines MG63 and osteoblast cell line hFOB1.19, as well as case matched normal (bone) or OS tissues (n = 3) using two primers. D. The expression of COL6A1 was detected by western blot and qRT-PCR upon treatment of C646 or A485 in OS cell lines, U2OS and Saos-2. E. The expression of COL6A1 was detected by western blot and qRT-PCR upon p300 transfection in OS cell lines, U2OS and Saos-2. F. ChIP assay demonstrated that H3K27 acetylation occurred in the promoter of COL6A1 in OS cell lines U2OS upon p300 transfection (left panel). ChIP-PCR assay confirmed the interaction between p300 and COL6A1 promoter, and this interaction did not exist at the IgG promoter, an internal control (right panel). G. The expression of COL6A1 was detected after co-transfection of c-Jun and p300 plasmids in U2OS cell line. H. The co-localization of c-Jun and p300 was confirmed by confocal microscopy (Scale bars: 50 µm). I. U2OS cells were co-transfected with different combinations of wild type (wt) and c-Jun-1-site-mutated reporter constructs (mt 1, mt 2, and mt 3), p300, c-Jun, and control. The relative luciferase activity was analyzed as previously described. Data represent the mean ± SD of 3 separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.