Fig. 2.

Loss of TNFR1 and SAM68 rescues cellular viability in BRCA2-depleted cancer cells. a Schematic overview of TNFR1 complex formation upon TNFα binding, leading to cell survival (complex I) or delayed caspase activation and cell death (complex II). b Flow cytometry analysis of KBM-7-shBRCA2 #2 cells, additionally carrying indicated shRNA vectors with IRES-driven mCherry cassettes. Cells were treated with doxycycline for 14 days and percentages of mCherry-positive cells were measured. c Indicated KBM-7-shBRCA2 cells carrying mCherry shRNA cassettes targeting TNFR1, SAM68 or a control sequence (‘SCR’) were treated with or without doxycycline to induce BRCA2 shRNA expression. Percentages of mCherry-positive cells were measured every 3 or 4 days for 3 weeks after start of doxycycline treatment. Ratios of mCherry-positive cells in doxycycline treated cultures vs. untreated cultures are indicated. Per condition, at least 30,000 events were measured. d BT-549 cells, stably transduced with pLKO.tet.shBRCA2 #2, were infected with IRES mCherry shRNA vectors as for b. Cells were treated with or without doxycycline, and percentages of mCherry-positive cells were measured. Ratios of mCherry-positive cells at indicated time points vs. mCherry-positive percentages at day 0 are indicated. Error bars indicate s.d. of three independent experiments. P values were calculated using two-tailed Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. e Representative flow cytometry plots of BT-549-shBRCA2 #2 cells from d are shown, carrying mCherry shRNA cassette for TNFR1 #2. Cells were treated for 15 days with or without doxycycline and gated based on mCherry positivity. Numbers indicate the percentages of mCherry-positive cells