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.