FIGURE 4.

Prdx6 hyperoxidation plays a role in H₂O₂-induced cellular toxicity. A, HeLa cells were treated with different concentrations of H₂O₂ as indicated for 20 min, washed with HBSS, and further incubated for 18 h. Cell lysates were subjected to immunoblotting with anti-Prdx6 SO₃H, anti-Prdx6, anti-p53, anti-p21, anti-cyclin B1, or anti-GAPDH antibodies. B, after treatment with different concentrations of H₂O₂ as described in A, cells were washed with HBSS, further incubated for 18 h, stained with propidium iodide as described under “Experimental Procedures”, analyzed with the FACSCalibur™ system, and then cell cycle distributions were determined with the Modfit LT 3.0 software. The results are expressed the mean ± S.D. for triplicate assays. C and D, HeLa cells were treated with different concentrations of H₂O₂ for 20 min, scraped into Eppendorf tubes, and centrifuged at 1000 × g for 5 min. Total PLA2 (C) and iPLA2 activities (D) were measured as described under “Experimental Procedures.” The results are expressed as mean ± S.D. for triplicate assays. E, HeLa cells were transiently transfected with wild-type Prdx6, the C47A mutant, the C32A mutant, and the C32A/C47A double mutant of Prdx6. At 36 h after transfection, cells were treated with or without 500 μm H₂O₂ for 20 min. Cell lysates were subjected to immunoblotting with anti-Prdx6 and anti-GAPDH antibodies, and iPLA2 activity was measured. F, model for the role of Prdx6 hyperoxidation in H₂O₂-induced cellular toxicity.