Suppression of c-FLIP sensitises breast cancer cell lines to TNF-Related Apoptosis Inducing Ligand (TRAIL). A, Cell lines were incubated with 20 ng/ml soluble TRAIL for 18 hours and cell viability assessed by cellTiter Blue viability assay. Viability is shown as a percentage of the untreated controls for each cell line (*: P < 0.01, n = 3). B, PKH67-stained cells pre-treated with c-FLIP siRNA and PKH26-stained cells pre-treated with control siRNA were mixed and co-cultured for 18 hours in complete media +/-20 ng/ml TRAIL, and assessed by flow cytometry for live/dead cells (see Supplementary figure S2). The percentage increase in cell death of cFLIP siRNA cells compared to their scrambled siRNA controls were plotted for each of the cell lines. TRAIL-treated co-cultures (white bars) and TRAIL-untreated co-cultures (Black bars) were plotted separately. Each co-culture was repeated in three independent experiments. + = P < 0.01, for percentage increase in cell death between c-FLIP siRNA and scrambled siRNA control. * = P < 0.01, for difference in c-FLIP siRNA mediated death between TRAIL-treated and untreated co-cultures. C, Cells were treated as in B and gated to give the percentage of remaining live cells after treatment. FT = c-FLIP siRNA.