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Fig. 2. Inhibition of GSK-3
blocks ceramide-induced apoptosis. (A) 10I cells were treated with 25 µM of C2-ceramide for 6 hours with or without the GSK-3 inhibitors LiCl (10 mM) and SB216763 (10 µM). Cell apoptosis was determined using DAPI staining and microscopy (to analyze nuclear fragmentation), and propidium iodide (PI) staining and flow cytometric analysis (to analyze DNA fragmentation). Arrowheads indicate apoptotic cells; percentages of apoptotic cells in the subG0 phase are marked in the histograms. (B) 10I cells were treated with 25 µM of C2-ceramide for 6 hours with or without various doses of LiCl and SB216763 as indicated. Cell apoptosis was detected using PI, annexin V and TUNEL staining, followed by flow cytometric analysis. The percentage of apoptotic cells is shown (mean ± s.d. of five replicates for PI staining and of triplicates for annexin V and TUNEL staining). Cells treated with DMSO were used as the reagent control. (C) 10I cells were transfected with 100 nM of GSK-3
siRNA (95.8% transfection efficiency) or 100 nM of GSK-3 siRNA (96.1% transfection efficiency) for 24 hours as described in Materials and Methods. After further treatment with 25 µM of C2-ceramide for 6 hours, the GSK-3, GSK-3, GS phosphorylated at serine 641 (P-GS), and levels of total GS (GS) were detected using western blotting. -actin levels were determined as an internal control. (D) Cells pre-transfected for 24 hours with control siRNA or GSK-3 siRNA (100 nM) were treated with 25 µM C2-ceramide for 6 hours. We used DAPI staining plus microscopy to determine cell apoptosis characterized by nuclear fragmentation, and PI staining plus flow cytometry to determine cell apoptosis characterized by DNA fragmentation. Arrowheads indicate the apoptotic cells; percentages of apoptotic cells in the subG0 phase are given in each micrograph.
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