NS1 suppressed of RhoA/pRb pathway. (A) A549 cells were transfected with pcDNA3.0 and pcDNA3.0-NS1 for 48 h and then analyzed for p21, cyclin D1, and p16INK4a protein expression levels by Western blotting. (B) Western blotting was used to detect the RhoA protein expression level. The data are shown as means ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (C) Western blotting for Rb and pRb protein. The data are shown as means ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (D) A549 cells were transfected with pcDNA3.0 and pcDNA3.0-NS1 for 48 h and then analyzed for CDK2, CDK4, and CDK6 protein expression levels by Western blotting. (E) A549 cells were cotransfected with pEZH2 and pcDNA3.0 or pcDNA3.0-NS1 for 48 h and then harvested. Chromatin proteins cross-linked to DNA were immunoprecipitated with IgG, E2F1, and DP-1. DNA that was pulled down was analyzed by PCR using primers that amplified the E2F/DP-1 binding region in the EZH2 promoter.