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Fig. 5. OXO-mediated cell-cell contact loss of RIE1-5 cells involves pS⁶⁴³PKC-dependent dynamic peripheral actin reorganization. (A,B) RIE1-5 cells (at 7080% confluence) on 10% FBS-DMEM-H-coated coverslips were treated with DMSO or 10 µM rottlerin for 24 hours (A), or with DMSO, 10 µM OXO alone or OXO plus rottlerin (10 µM OXO + 10 µM rottlerin) for 24 hours (B). Cells were then stained for actin (A) or immunostained for -catenin (B), as explained in Materials and Methods. (C-E) Cells were infected with adenovirus for GFP (Ad-cont), dominant negative K376A PKC (Ad-DN PKC) or K368R PKC (Ad-DN PKC), or wild-type PKC (Ad-WT PKC) or PKC (Ad-WT PKC) for 8 hours. After infection, media were replaced with fresh culture media. DMSO or 10 µM OXO was then treated for 24 hours, prior to immunofluorescent staining against ZO1 (left panels) or -catenin (right panels; C,D), or stained for actin using phalloidin-conjugated Rhodamine (E). (F) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Con), K376A DN PKC (DN), or WT PKC (WT), for 8 hours. After the viruses had been washed out, cells were treated with DMSO or 10 µM OXO for 24 hours, before preparation of whole cell lysates. An equal amount of proteins was subjected to standard western blotting for the indicated molecules. (G) RIE1-4 cells were manipulated as in (C) prior to ZO1 immunofluorescent staining. Data shown are representative of three independent experiments.

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