Fig 2: A mild cFLIP knocking down of FHL124 cells manifested with an increased apoptotic phenotype.A,B Scrambled control shRNA FHL124 cells showed normal nuclear morphology 24 h after TNFa and CHX treatment. C,D KD3 cFLIP knockdown FHL124 cells demonstrated scattered apoptotic cells after 7 h of treatment. E,F KD3 cFLIP knockdown FHL124 cells demonstrated a large volume of apoptotic cells after 24 h of treatment. G,H Active apoptotic cells were analyzed by APC-Annexin V and 7-AAD stain and flow cytometry in scrambled shRNA FHL124 control cells treated with or without 24 h TNFa and CHX stimulation. J,K Active apoptotic cells were analyzed by APC-Annexin V and 7-AAD stain and flow cytometry in KD3 cFLIP knockdown FHL124 cells treated with or without 24 h TNFa and CHX stimulation. L Quantitative data of the ratio of apoptosis in control and cFLIP knockdown FHL124 cells. One-way ANOVA with Tukey’s Honest post-hoc analysis was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 3: cFLIP is highly expressed in FHL124 cells and also significantly upregulated after TNFa stimulation.A Anti-apoptotic protein expression in FHL124 and HeLa cell with and without TNFa and TNFa plus CHX stimulation. B Relative cFLIP mRNA expression in FHL124 and HeLa cells with and without TNFa and CHX stimulation. FHL 124 cells demonstrated a more robust response to TNFa and CHX stimulation compared to HeLa cells. C The endogenous cFLIP, Caspase-8, caspase-9, and caspase-3 mRNA levels in FHL124 cells vs. HeLa cells. A much higher cFLIP mRNA expression was seen in FHL124 cells compared to HeLa cells. D,E cFLIP protein expression after TNFa and CHX stimulation in FHL124 and HeLa cells. cFLIP protein level was increased around 3-fold in FHL124 cells at 5 h after TNFa and CHX stimulation, while only a mild increase was seen in HeLa cells after stimulation. F,G FHL124 cells had 6-fold endogenous cFLIP protein levels vs. HeLa cells. KD3 shRNA was able to knock down 50% of cFLIP expression in FHL124 cells. Each assay was repeated at least three times. One-way ANOVA with Tukey’s Honest post-hoc analysis was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 4: Proposed Model of TRAIL-Susceptiblity in Breast Tumour Subpopulations: (a) Epithelial-like cells have cytoplasmic cFLIP and consequently are resistant to TRAIL-mediated apoptosis. (b) Mesenchymal-like cells have comparatively reduced cytoplasmic cFLIP and elevated nuclear cFLIP due to its role in the relatively more active Wnt pathway, and are consequently TRAIL sensitive (c) The consequences of cFLIP inhibition: sensitisation to TRAIL, reduction in Wnt signalling and potentially a reduction in bCSC self-renewal and proliferation

Fig 5: 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)