Extended Data Fig. 8: NIK−/− and SIX1−/−SIX2−/− fibroblasts are sensitized to BV6–TNF-induced cell death.
From: A NIK–SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB
We validated the observation that SIX1−/−SIX2−/− sensitized NSCLC cells to cell death induced by BV6 and TNF in SV40 immortalized STAT1−/− fibroblasts. a, b, Fibroblasts of the indicated genotype were treated with BV6 (a) or TNF (b). Cell viability was determined by measuring ATP after 24 h. Cell survival rate was normalized to each genotype’s untreated control. Neither BV6 nor TNF (10 ng ml−1) alone induced fibroblast cell death. c, NIK−/− and SIX1−/−SIX2−/− fibroblasts are sensitized to BV6–TNF-induced cell death. Left, experiments and analysis as in a. Right, representative images showing the cell death phenotype induced by BV6–TNF in fibroblasts of the indicated genotypes. Scale bars represent 50 μm. d, Time course of combined BV6 (2.5 μM) and TNF (25 ng ml−1) treatment. NIK−/− and SIX1−/−SIX2−/− fibroblasts exhibited increased cleavage of poly ADP-ribose polymerase (PARP) and caspase-3 in BV6–TNF-treated fibroblasts. BV6–TNF induced NIK-dependent expression of both SIX1 and SIX2, suggesting that this cascade may be responsible for resistance to this treatment. e, f, We introduced a silent mutation in the gRNA recognition sequence of SIX2 cDNA that cannot be targeted by CRISPR–Cas9 (SIX2R, e, top). Expression of SIX2R in SIX1−/−SIX2−/− fibroblasts rescued the cell death phenotype (e) and suppressed both PARP and caspase-3 cleavage (f) induced by BV6–TNF. Wild-type and SIX1−/−SIX2−/− fibroblasts were transduced with Fluc or SIX2R lentivirus. After 72 h, cells were mock-treated or treated with 0.2 μM BV6 plus 10 ng ml−1 TNF for 24 h. Cell viability was determined by measuring ATP. Cell survival was normalized to each untreated control (e, bottom). For western blot, cells were treated with 2.5 μM BV6 plus 25 ng ml−1 TNF for 6 h (f). All quantified data are mean ± s.d. from nine independent experiments. **P < 0.01, ****P < 0.0001. Western blot data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.
