Figure 4.

Silenced B‐myb induced cell premature senescence through the ROS/p53/p21 pathway. A, HAECs were per‐incubated with PFTα (3 μmol/L) for 60 min followed by transfected with siB‐myb or siNC in the presence of PFTα (3 μmol/L) for 7 d. The expression levels of B‐myb, p‐p53, p53 and p21 proteins were analysed by western blotting. GAPDH was used as a loading control. A typical group of blots is shown from one of three independent experiments. B, Cells were incubated with PFTα (3 μmol/L) or NAC (5 mmol/L) for 60 min followed by transfection with siB‐myb in the presence of PFTα (3 μmol/L) or NAC (5 mmol/L) for 7 d. The cells were then stained with SA‐β‐gal. A representative group image of stained cultures is shown (×100 magnification). The percentage rate of SA‐β‐gal‐positive cells was analysed. Data are presented as mean ± SEM of three independent experiments. *** indicates P<.001 compared between two groups. C, Cells were per‐incubated with NAC (5 mmol/L) for 60 min followed by transfection with siB‐myb in the presence of NAC (5 mmol/L) for 7 d. The production of intracellular ROS in the cells was determined with ROS indicator DCFH‐DA. A representative group image of stained cultures is shown (×100 magnification). The percentage rate of ROS‐positive cells was analysed. Data are presented as mean ± SEM of three independent experiments. *** indicates P<.001 compared between two groups. D, Cells were incubated with or without NAC (5 mmol/L) for 60 min followed by transfection with siB‐myb or siNC in the presence of NAC (5 mmol/L) for 7 d. The expression levels of B‐myb, p‐p53, p53 and p21 protein in the cell were analysed by western blotting. GAPDH was used as a loading control. A typical group of blots is shown from one of three independent experiments