FIGURE 1.

H₂O₂ disrupts phosphoinositide homeostasis and the actin cytoskeleton. Cells were treated without (Control) or with H₂O₂ for 20 min unless otherwise indicated. A, TLC. HeLa cells were labeled with [³²P]orthophosphate and exposed to H₂O₂. ³²P-Labeled lipids were analyzed by TLC and PhosphorImager analysis. Left, a typical fluorogram of ³²P-labeled lipids after separation by TLC; right, quantitation of ³²P-labeled lipids (mean ± S.E., n = 3). Amounts of [³²P]PIP₂ and PIP were expressed as a percentage of the total labeled phospholipids. Asterisks denote statistically significant compared with control, with p < 0.05, in this and all other panels in this figure. B, HPLC. HeLa cells were exposed to 0 or 0.5 mm H₂O₂ and lipids were extracted. Phospholipids were deacylated and negatively charged glycerol head groups were eluted and detected with suppressed conductivity (μS, microsiemen units). Left, HPLC elution profiles; right, quantitation (mean ± S.E., n = 3). C, actin cytoskeleton. COS cells were fixed and stained with FITC-phalloidin to detect polymerized actin fibers. Data shown are representative of two independent experiments. Left, representative images; right, scoring of actin morphology in cells from 10 randomly chosen fields per condition in a blinded fashion. The percentage of cells with normal long or abnormal actin stress fibers were plotted. 40–50 cells were analyzed per condition. D, partitioning of actin in Triton-soluble and -insoluble fractions. HSS, high speed supernatant. Left, Western blot of a representative experiment. Two samples from each condition are shown, and the amount of high speed supernatant loaded is half as much as that in the LSP and HSP; right, ratios of actin in HSP/LSP fractions. Values are mean ± S.E., n = 3. Error bars indicate S.E.M.