Fig. 3

Cytoplasmic but not nuclear cFLIP protects against TRAIL-mediated cell death (a) Western blots for cFLIP performed on cytoplasmic and nuclear protein extracts of MCF-7 s transfected with overexpression constructs; mock (M, empty vector control), cytoplasmic-localised cFLIP (C) and nuclear-localised cFLIP (N) (loading controls = α-tubulin and lamin A/C) (b) Densitometry analysis of Western blots for cFLIP performed on cytoplasmic and nuclear protein extracts of MCF-7 s expressing mutant cFLIP. Data is average of 3 independent protein samples normalised to Mock control. (c) Tumoursphere Assay of MCF-7 cells stably transfected with either mock, cytoplasmic-localised cFLIP or nuclear-localised cFLIP in the presence (T) or absence (−) of 20 ng/ml TRAIL. The percentage of tumourspheres is shown relative to the number of cells seeded (d) Immunofluorescence of MCF-7 cells cultured in the presence or absence of 0.1 ng/ml leptomycin-B (LMB1) for 24 h (cFLIP = grey, DAPI = red) (e) Cell Titre Blue Assay of MCF-7 cells cultured for 24 h in the presence or absence of 0.1 ng/ml LMB1 before 18 h treatment with 20 ng/ml TRAIL. Cell viability is shown as relative to the untreated control. (f) Tumoursphere Assay in the presence (T) or absence (−) of 20 ng/ml TRAIL, of MCF-7 cells cultured in adherent conditions for 24 h in the presence or absence of 0.1 ng/ml LMB1. The percentage of tumourspheres is shown relative to the number of cells seeded. (*p < 0.05, paired t-test, error bars = SEM; all graphs represent means of at least 3 independent experiments)