Figure 2.

The effect of oxidative stress on bmi1, p16^INK4a, and p21^WAF1/Cip expression in BECs. A: bmi1 mRNA expression was significantly lower in BECs on days 2, 3, and 4 after H₂O₂ (112.5 μmol/L, 2 hours) treatment. P16^INK4a mRNA expression was increased significantly on days 3, 4, and 5 after H₂O₂ treatment. P21^WAF1/Cip mRNA expression was high on day 1, gradually decreased on days 2, 3, and 4 after H₂O₂ treatment. Expression of mRNA was quantified with real-time PCR. The expression was normalized as a ratio using GAPDH as a housekeeping gene. Data are expressed as the mean ± SD. *P < 0.01, **P < 0.05 compared to the control, n = 3 for each group. B: Immunoblot analysis for bmi1, p16^INK4a, and p21^WAF1/Cip protein expression in BECs treated with H₂O₂ (112.5 μmol/L, 2 hours) and control BECs. A pool of protein samples prepared in three independent experiments was used and analysis was performed twice. C: Intensity of each band in immunoblot analysis (B) was quantified by densitometry, normalized as a ratio using α-tubulin as an internal control, and statistically analyzed. Bmi1 protein expression was significantly lower in BECs on days 3, 4, and 5 after H₂O₂ treatment. P16^INK4a protein expression was significantly high after H₂O₂ treatment. P21^WAF1/Cip protein expression was high on day 2 after H₂O₂ treatment and normalized thereafter. *P < 0.01, **P < 0.05 compared to control.