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Fig. 7. Cell death induced by active GSK-3 is partially rescued by expression of aPKC. (A) Wild-type MDCK and GFP-aPKC cells were cultured on Matrigel (72 hours) and infected with Ad-HA-GSK-3 (S9A). Immunostaining analysis was performed with GFP (green), cleaved caspase-3 (red) and HA (white). (B) Wild-type MDCK and GFP-aPKC cells were cultured on Matrigel (72 hours) and infected with Ad-gal or Ad-HA-GSK-3 (S9A). The percentage of cysts containing apoptotic cells was quantified by cleaved caspase-3 staining. Results are the mean±s.d. of three experiments. (C) MDCK cysts grown in collagen were treated with either the non-myristoylated (control) or myristoylated form of aPKC pseudosubstrate peptide at 50 µM at day 4; aPKC pseudosubstrate-containing media were refreshed everyday. Immunostaining with aPKC (green) and phosphorylated GSK-3 (red) was performed at day 7. (D) Wild-type MDCK and GFP-aPKC-overexpressing MDCK cells were embedded into collagen and cultured for 7 days. Immunostaining with GFP-aPKC (green) and phosphorylated GSK-3 (white) was performed at day 7. Bars, 10 µm. (E) Wild-type MDCK and PAR6N lysates at day 7 were subjected to immunoblot of total- and phospho-JNK antibody.

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