Figure 1.
iNOS exerts distinct functions during TNF/IFNγ-induced death of diverse cell lines. (A) Murine BMDMs were treated with TNF/IFNγ for 24 h. Protein expression of iNOS was analyzed by immunoblotting (n = 3). (B) Murine BMDMs were treated with TNF/IFNγ in the presence or absence of the iNOS inhibitor 1400W (100 μM) for 24 h (n = 3). Nitrite (NO2−) production in the culture medium was measured by the Griess assay. Two-tailed t test was performed: ****P < 0.0001. (C) Representative images of cell death in murine BMDMs after 48 h treatments of TNF/IFNγ in the presence or absence of 1400W. PI staining was used to show dead cells. Scale bar, 275 μm. (D) Murine BMDMs were treated with TNF/IFNγ in the presence or absence of 1400W for 48 h (n = 3). Cell death was measured by PI staining followed by flow cytometry. Two-tailed t test was performed: ****P < 0.0001. (E and F) HCT-116 and HT-29 cells (E) and HeLa and THP-1 cells (F) were treated as indicated for 24 h. Protein expression of iNOS was analyzed by immunoblotting (n = 4). (G) HeLa, THP-1, HCT-116, and HT-29 cells were treated with TNF/IFNγ for 24 h (n = 3). Nitrite (NO2−) production in the culture medium was measured. Two-tailed t test was performed: NS (not significant). (H and I) HeLa (H) or THP-1 (I) cells were treated with TNF/IFNγ in the presence or absence of 1,400 W, or the NOS inhibitor L-NAME (1 mM) for 48 h (n = 3). Cell death was measured by LDH levels released in the culture medium. One-way analysis of variance (ANOVA) was performed: NS. (J) HT-29 cells were treated with TNF/IFNγ in the presence or absence of IL-1β (100 ng/ml) for 48 h (n = 3). Cell death was measured by PI staining followed by flow cytometry. A two-tailed t test was performed: NS. (K) EMT6 cells were treated with TNF/IFNγ for indicated time points. Protein expression of iNOS was analyzed by immunoblotting (n = 2). (L) Representative images of PI staining in EMT6 cells after 36 h treatments of TNF/IFNγ in the presence or absence of 1400W; scale bar, 275 μm. (M) Parental EMT6 and NOS2 KO cells (#1 and #2 clones with different gRNA) were treated with TNF/IFNγ for 36 h (n = 3). Cell death was measured by PI staining followed by quantification. One-way ANOVA was performed: NS; **P = 0.0036. 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 F1.

iNOS exerts distinct functions during TNF/IFNγ-induced death of diverse cell lines. (A) Murine BMDMs were treated with TNF/IFNγ for 24 h. Protein expression of iNOS was analyzed by immunoblotting (n = 3). (B) Murine BMDMs were treated with TNF/IFNγ in the presence or absence of the iNOS inhibitor 1400W (100 μM) for 24 h (n = 3). Nitrite (NO2−) production in the culture medium was measured by the Griess assay. Two-tailed t test was performed: ****P < 0.0001. (C) Representative images of cell death in murine BMDMs after 48 h treatments of TNF/IFNγ in the presence or absence of 1400W. PI staining was used to show dead cells. Scale bar, 275 μm. (D) Murine BMDMs were treated with TNF/IFNγ in the presence or absence of 1400W for 48 h (n = 3). Cell death was measured by PI staining followed by flow cytometry. Two-tailed t test was performed: ****P < 0.0001. (E and F) HCT-116 and HT-29 cells (E) and HeLa and THP-1 cells (F) were treated as indicated for 24 h. Protein expression of iNOS was analyzed by immunoblotting (n = 4). (G) HeLa, THP-1, HCT-116, and HT-29 cells were treated with TNF/IFNγ for 24 h (n = 3). Nitrite (NO2−) production in the culture medium was measured. Two-tailed t test was performed: NS (not significant). (H and I) HeLa (H) or THP-1 (I) cells were treated with TNF/IFNγ in the presence or absence of 1,400 W, or the NOS inhibitor L-NAME (1 mM) for 48 h (n = 3). Cell death was measured by LDH levels released in the culture medium. One-way analysis of variance (ANOVA) was performed: NS. (J) HT-29 cells were treated with TNF/IFNγ in the presence or absence of IL-1β (100 ng/ml) for 48 h (n = 3). Cell death was measured by PI staining followed by flow cytometry. A two-tailed t test was performed: NS. (K) EMT6 cells were treated with TNF/IFNγ for indicated time points. Protein expression of iNOS was analyzed by immunoblotting (n = 2). (L) Representative images of PI staining in EMT6 cells after 36 h treatments of TNF/IFNγ in the presence or absence of 1400W; scale bar, 275 μm. (M) Parental EMT6 and NOS2 KO cells (#1 and #2 clones with different gRNA) were treated with TNF/IFNγ for 36 h (n = 3). Cell death was measured by PI staining followed by quantification. One-way ANOVA was performed: NS; **P = 0.0036. 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 F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal