Fig. 4.
Quantitative changes in polycomb proteins and demethylases in E6/E7 cells and effect of H3K27me3 loss on HOX expression. (A) Western blot analysis of PRC2 core proteins and BMI1 (PRC1) in 60-μg total cell lysates from E6/E7 cells and pBabe cells. The blots show increased expression of EZH2 and downregulation of BMI1 but no significant change in SUZ12 or EED isoform levels in E6/E7 cells compared to pBabe cells. Increased expression of KDM6A was also observed in E6/E7 cells, while KDM6B protein levels remained very low in both cell lines. (B) QRT-PCR analysis of H3K27me3-targeted HOX gene expression in cycling E6/E7 cells compared to pBabe cells. Expression data are presented as CT values normalized to β2-M, and fold changes, calculated using the 2−ΔΔCT method, are indicated at the bottom. Data represent three independent experiments. *, P < 0.05; **, P < 0.001. (C) ChIP-PCR analysis showing a reduction in H3K27me3 at targeted HOX promoter fragments between cycling pBabe controls and E6/E7-expressing cells. Data are representative of two independent ChIP-PCR experiments.