Figure 1. Nrf2 was Required for the p21-Dependent Cellular Protection in Response to Oxidative Stress.

(A) p21 exhibited antioxidant functions. HCT116-p21^+/+, HCT116-p21^-/-, and HCT116-p21^-/- cells transfected with an expression vector for p21, were treated with the indicated doses of H₂O₂ for 12 hrs. Intracellular ROS levels were measured by the dichlorofluorescein/flow cytometry method. The representative histograms of ROS analysis in HCT116-p21^+/+cells and HCT116-p21^-/- cells are shown in the upper two panels. (B) HCT116-p21^+/+ cells with knockdown of Nrf2 had higher intracellular levels of ROS. HCT116-p21^+/+ cells were transfected with either control siRNA or Nrf2-siRNA. At 72 hrs post-transfection, levels of Nrf2 and intracellular ROS were measured. (C) Ectopic expression of p21 enhanced cell survival in response to H₂O₂ in MEF-Nrf2^+/+, but not in MEF-Nrf2^-/- cells. MEF-Nrf2^+/+ or MEF-Nrf2^-/- cells were cotransfected with the indicated expression vectors for 24 hrs. GFP was used to mark the transfected cells. Following treatment with H₂O₂, over 150 GFP positive cells were counted for both surviving and apoptotic cells by fluorescence microscopy. The percentage of surviving cells was plotted. (D) Knockdown of p21 sensitized cells to H₂O₂-induced cell death in MEF-Nrf2^+/+, but not in MEF-Nrf2^-/- cells. Cells were transfected with p21-siRNA or control siRNA for 72 hrs before treatment with H₂O₂. Apoptotic cells were detected using the Annexin V-FITC/flow cytometry method. All error bars indicate standard deviations calculated from three independent experiments. *p<0.05 compared with its control.