Induction of HBA expression after siRNA-mediated depletion of EZH2 in nonerythroid cells. HeLa cells were transfected with either control siRNA or EZH2-specific siRNA and were harvested every 2 days over a 6-day time course as shown. (A) Real time RT-PCR analysis of EZH2, HBA2, STEAP1, and HBB expression. Results are shown as either the percentage of EZH2 signal remaining (top panel) or the fold change (bottom panels) in the EZH2 siRNA-treated population relative to the control siRNA–treated population after normalizing for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. (B) Protein extracts from a representative time course analyzed by Western blot using antibodies against EZH2 (top panel) or RNA polymerase II as loading control (bottom panel). (C) Chromatin was harvested after 6 days with the indicated siRNA and was analyzed by quantitative ChIP using an antibody against H3K27me3. Material was analyzed by real-time PCR at amplicons 53 (background enrichment only), HBA ex1-2 and HBA ex3 from the α-globin genes (see Figure 1) and the CpG-island of the STEAP1 gene. Data are presented as either raw enrichment (the percentage of input material precipitated at each amplicon; top panel) or the percentage of signal remaining in the EZH2 siRNA-treated cells relative to control siRNA–treated cells (bottom panel). Data in panels A and C show the mean values obtained from replicate time courses with error bars indicating the range of values.