Fig. 4

Nuclear cFLIP promotes Wnt-signalling (a) Western blotting for cFLIP and beta-catenin performed on cytoplasmic protein extracts of MCF-7 and MDA-MB-231 cell lines transfected with siRNA targeting cFLIP or a scrambled control (loading control alpha-tubulin) (b) Immunofluorescence for cFLIP and beta catenin of MDA-MB-231 cells transfected with siRNA targeting cFLIP or a scrambled control (red = cFLIP, green = beta catenin, blue = DAPI) (c) qPCR analysis for mRNA levels of cFLIP and Axin2 in MCF-7 and MDA-MB-231 cells transfected with siRNA targeting cFLIP or a scrambled control and cultured in the presence of 10 ng/ml Wnt3a for 48 h. Gene expression is expressed as relative to the untreated control. (d) TOPFlash luciferase reporter assay of MCF-7 cells transfected with overexpression constructs; mock (empty), cytoplasmic-localised cFLIP and nuclear-localised cFLIP and cultured in the presence of 10 ng/ml Wnt3a for 48 h. Luciferase output is expressed as relative to that of the mock-transfected cells. (*p < 0.05, paired t-test, error bars = SEM; all graphs represent means of at least 3 independent experiments)