Fig. 2.

Increased expression of EZH2 in differentiating E6/E7 cells. (A) QRT-PCR of EZH2 in differentiated pBabe and E6/E7 cells following CaCl₂-induced differentiation. Data represent transcripts at 48 h of differentiation for three independent experiments. (B) Using immunohistochemistry detection, the EZH2 protein was shown to be confined to the nucleus in the basal cells of HFK rafts, while more abundant and stronger nuclear staining was observed throughout the epithelial layers of E6/E7 raft sections. Higher levels of P-EZH2-Ser21 were observed in the nuclei of E6/E7 cells than in normal HFKs. Control sections of pBabe and E6/E7 raft sections showed negative nuclei and minimal background staining following incubation of the P-EZH2-Ser21 antibody with a 3-fold excess of blocking peptide (EZH2-BP). (C) Quantification by two observers of the median number of cells positive for EZH2 compared to Ki67 in 10 independent frames from raft sections indicated that EZH2 was expressed at a higher level in E6/E7 cells than in normal HFKs. (D) Western blot showing that E6/E7 cells contain high levels of active Akt compared to pBabe cells. (E) siRNA knockdown of Akt, measured by protein levels and reduced phosphorylation of the Akt substrate GSK-3β, results in a reduced level of P-EZH2-Ser21. (F) Pharmacological reduction of P-Akt results in reduced levels of P-Akt in E6/E7 cells and in a significant increase in H3K27me23 levels in E6/E7 cells compared to pBabe controls.