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Fig. 4. Differential cofilin phosphorylation and peripheral actin reorganization in RIE1-5 and RIE1-5/1 cells. (A) Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. After cells had adhered and spread typically, they were treated with either DMSO or 30 µM OXO for 30 minutes. Cells were then processed for actin staining using phalloidin-conjugated with Rhodamine, as described in Materials and Methods. Note that 30 µM OXO treatment disrupted actin filament organization only in RIE1-5 cells. (B) Cells on normal culture medium-coated coverslips were incubated with 20 µM DCHF-PBS for 30 minutes, prior to washing and visualization of ROS-positive cells by fluorescent microscopy. (C,D) Cells (at 7080% confluence) in 60 mm culture dishes were not pretreated or pretreated with the indicated pharmacological inhibitors, as in Fig. 3, prior to treatment with DMSO or 10 µM OXO for 24 hours (C) or with DMSO (i.e. 0 µM of cytochalasin D) or cytochalasin D at the indicated concentrations for 1 hour (D). Whole cell lysates were then prepared and normalized protein amounts were used in standard western blotting for the indicated molecules. Note that RIE1-5/1 cells showed more sustained pS³cofilin levels than RIE1-5 cells did, even after treatment with cytochalasin D. (E) Cells on normal culture medium-coated coverslips were treated with DMSO or cytochalasin D (0.05 or 0.2 µM) for 1 hour, prior to actin staining as in A. (F) Cells were manipulated as described in the legend of Fig. 3, with the exception of staining for actin using phalloidin-conjugated with Rhodamine. Data shown are representative of three independent experiments.

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