Figure 3. Depletion of EZH2 leads to re-expression of p27^Kip1 in pancreatic cancer cells.

MIA-PaCa2 and Panc04.03 pancreatic cancer cells were transfected with a control vector (shVector) or the shEZH2 silencing vector (shEZH2). A, 48 hours post-transfection, the cell pellet was collected, mRNA was obtained and RT-PCR was performed as described in Materials and Methods. Increased expression of MYT1, a known EZH2 target gene, was used as a positive control. B, 48 hours post-transfection, the cell pellet was collected, protein was obtained. Cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to indicated proteins. C, 48 hours post-transfection, Panc04.03 cell pellet was collected. Panc04.03 cancer cells were treated with DMSO (Control) or MG132 (10 μmol/L) for 6 hours. Cell lysates were prepared, separated by SDS-PAGE (50μg/well), transferred to PVDF membrane, and immunoblotted as indicated. An increase of IκBα expression was used as a positive marker of MG132 treatment. D, Binding of EZH2 to the promoter or exon 1 of p27^Kip1 gene was assayed in Panc04.03 cancer cells by ChIP. E, Panc04.03 cancer cells were transfected with a control vector (shVector) or the shEZH2 silencing vector (shEZH2). 48 hours post-transfection, genomic chromatin fragments were immunoprecipitated with trimethyl-H3-K27 and acetyl-H3-K14 at the p27^Kip1 promoter or exon 1. PCR analysis on input chromatin (first 2 lanes) confirmed that equal chromatin amounts were used for ChIP. (F, G) Immunohistochemical staining of a pair of serial sections from the same pancreatic tumor demonstrates nuclear localization of EZH2 (F) and loss of p27^Kip1 expression (G) in the same cancer cells, whereas absence of EZH2 nuclear staining, but p27^Kip1 nuclear accumulation has been observed in benign pancreatic lesions (G, F). Pancreatic intraepithelial neoplasia (PanIN).