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. 2022 Feb 6;11(3):563. doi: 10.3390/cells11030563

Figure 1.

Figure 1

IFNγ preferentially promotes cell death and the release of LDH induced by various necroptosis inducers in primary AECs. Mouse primary AECs were incubated with LPS (100 ng/mL), IFNα2 (50 ng/mL), IFNγ (50 ng/mL), poly(I:C) (PIC, 0.5 μg/mL), CL075 (1 μg/mL), ODN1585 (1 μM), IL-4 (15 ng/mL), or IL-10 (15 ng/mL) for 24 h, then treated for 6 h with TNFα (15 ng/mL) plus 100 nM Smac mimetic AZD5582 and 25 µM z-VAD-FMK (TSZ), poly(I:C) (0.2 µg/mL) plus 100 nM Smac mimetic AZD5582 and 40 µM z-VAD-FMK (PSZ), or LPS (200 ng/mL) plus 100 nM Smac mimetic AZD5582 and 25 µM z-VAD-FMK (LSZ). (A) AEC viability was assessed by MTS assay using CellTiter AQueous reagent and cell survival rates were calculated by comparison to individual control groups without treatment of necroptosis inducers and are presented as means ± SE. Con.+TSZ or PSZ, n = 12; IFNγ+TSZ or PSZ, n = 10; all other treatments, n = 4–8. ** p < 0.01; *** p < 0.001 versus control (Con.) in each treatment group. NS, no significance. (B) LDH activities in cell supernatant were determined. Relative changes in LDH are presented as fold ± SE of individual control groups without treatment of necroptosis inducers. Con. or IFNγ+TSZ, n = 10; Con. or IFNγ+PSZ, n = 12; all other treatments, n = 4–7. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (Con.) in each group. NS, no significance. (C) Mouse AECs were incubated without or with IFNγ (50 ng/mL) in the presence of DMSO or JAK kinase inhibitor-1 (JAKi, 1 μM) for 24 h, washed and then treated for 6 h with necroptosis inducers TSZ or PSZ in the presence of DMSO, GSK872 (5 μM) or JAKi (1 μM). LDH activities in cell supernatant were determined and the relative changes are presented as fold ± SE of individual control groups without treatment of necroptosis inducers. TSZ group: DMSO or DMSO+IFNγ, n = 7; others, n = 3–4. PSZ group: DMSO or DMSO+IFNγ, n = 9; others, n = 5. *** p < 0.001 versus DMSO in each group. ### p < 0.001 versus DMSO+IFNγ in each group. NS, no significance. One-way ANOVA was performed in (A–C).