Fig. 4.

Atg5 regulates H₂O₂-induced senescence in part via upregulation of p21. (A) and (B) WI38 cells were transfected with control siRNA (CTL siRNA) or p21 siRNA as described in Materials and methods. Three days after the transfection, they were exposed to 250 μM H₂O₂ for 2 h. Cells with CTL siRNA transfection but without H₂O₂ treatment were included as a control. Six days after H₂O₂ treatment, they were harvested for the analyses of p21 expression by Western blots and senescence induction by BrdU and SA-β-gal stainings. (A) A representative Western blot image of p21 is shown. The β-actin blot is included as a loading control. (B) Percentage of BrdU and SA-β-gal positive cells are shown. The data are presented in (B) as means±SD from three independent experiments. **P ≤ 0.01. (C) and (D) WI38 cells were transfected with vehicle, control siRNA (CTL siRNA) or Atg5 siRNA as described above. Three days after of the transfection, they were exposed to 250 μM H₂O₂ for 2 h. Cells without siRNA transfection and H₂O₂ treatment were included as a control (CTL). Six days after H₂O₂ treatment, they were harvested for the analyses of p21 expression by Western blot and qRT-PCR. (C) A representative Western blot image of p21 is shown. The β-actin blot is included as a loading control. (D) The relative ratios of p21 mRNA ΔCT values between the CTL and H₂O₂-treated cells are presented as means±SD (n=3).