Figure 6.

IFNγ/LPS-induced iNOS sensitizes macrophages to caspase-8 and BAX/BAK-mediated death

(A) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) for 2–24 h. Nitrite (NO₂⁻) production and iNOS expression were measured by the Griess assay and immunoblot (n = 3).

(B and C) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) ± the iNOS inhibitor 1400W (10 μM) or DMSO for 24 h. (B) Nitrite (NO₂⁻) production and cell death were measured by the Griess assay and (C) PI exclusion as measured by flow cytometry (n = 4).

(D) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) ± 1400W (10 μM) for 24 h. The nitric oxide donor SNAP (200 μM) or DMSO were provided 8 h post-treatment with LPS. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 5).

(E) WT BMDMs were treated with 1400W (10 μM) ± IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) or TNF (100 ng/mL) for 24 h. SNAP (200 μM) or DMSO were added to cells 8 h post-treatment with LPS or TNF. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4).

(F, G, and H) WT or Nos2^−/− BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) or Pam-3-CSK4 (P3C, 500 ng/mL) for 16, 24, or 48 h. ABT-737 (1 μM) and cycloheximide (CHX, 10 μg/mL) treatment for (G) 6 h or (H) 2 h was used as a positive control. (F) Cell death and (G) cytochrome c retention was assessed by PI exclusion as measured by flow cytometry or intracellular cytochrome c staining and flow cytometric analysis (n = 6). (H) Cell death pathway activation was assessed by immunoblot of cell supernatants (S/N) and cell lysates (n = 2).

Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p ≤ 0.05 (^∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figure S6 and S7.