iNOS is dispensable in TNF/IFNγ-induced human and murine origin cancer cell death. (A) Representative images of cell death in indicated cancer cell lines with TNF, IFNγ, or TNF/IFNγ treatments for 36 h (n = 3). PI staining was used to show dead cells. Scale bar, 275 μm. (B) Representative images of PI staining in HeLa cells after 36 h treatment with TNF alone or plus indicated concentrations of IFNγ. Scale bar, 275 μm. (C) HeLa cells were treated with TNF/IFNγ in the presence or absence of 1400W, or L-NAME for 48 h (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: NS. (D) EMT6 cells (parental and Nos2 KO clones #1 and #2 with different gRNA) were treated with IFNγ for 12 h. Protein expression of iNOS was analyzed by immunoblotting (n = 3). (E) HeLa cells were treated with TNF, IFNγ, or TNF/IFNγ for 36 h. The expression of cleaved and full-length caspase-3/7/8 and GSDME proteins were analyzed by immunoblotting (n = 3). (F and G) Schematic of the biochemical mechanism of TNF or TLR ligands plus IFNγ induced cell death in murine BMDMs (F) or cancer cells (G). Asterisk: TNF/IFNγ failed to induce the expression of iNOS in human cancer cells, or the induced iNOS in murine cancer cells has no effect on cell death. Data represent the mean value ± SD from n biological replicates, or a representative immunoblot from n independent experiments. Source data are available for this figure: SourceData FS1.