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. 2011 Dec 22;2(12):e245. doi: 10.1038/cddis.2011.131

Figure 3.

Figure 3

Overexpression of c-FLIP isoforms protects urothelial carcinoma cells against apoptosis. (a) VMCub1 and SD cells were treated with 10 μg/ml CHX for up to 24 h. c-FLIP protein expression was determined by western blotting. β-Actin served as a loading control. (b) Original pEF-vectors and the generated pIRES2EGFP-c-FLIP constructs were transiently transfected into 293T cells. The c-FLIP and GFP protein expression was analysed by western blotting with tubulin as the loading control. (c–f) pIRES2EGFP-c-FLIP constructs were transiently overexpressed in VMCub1 cells (c and d) or SD cells (e and f). The urothelial carcinoma cells were left untreated or stimulated with 1 ng/ml CD95L for 4 h. Sensitivity to apoptosis in transfected cells was analysed by flow cytometry. Intracellular staining of active caspase-3 was used as a marker for apoptotic cells and the transfected cells were identified by GFP expression. (c and e) Histograms are representative for three independent experiments with percentages shown for stimulated samples. (d and f) Data are shown as the percentages within the GFP-positive population with the mean of three independent experiments ±S.D.