Figure 2.

Both extrinsic caspase-8 and the mitochondrial apoptosis effector proteins BAX/BAK contribute to IFNγ/LPS-induced macrophage killing

(A) WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 24 or 48 h. Treatment with LPS and Compound A (Cp. A, 1 μM) (extrinsic apoptosis) or LPS and Z-VAD-fmk (Z-VAD, 20 μM) (necroptosis) for 24 h were used as controls. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4).

(B) Immunoblot analysis of WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 8, 12 or 24 h. WT BMDMs were treated with LPS and Cp. A for 12 h as a control (n = 3).

(C) WT and Bax^−/−Bak^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 24 or 48 h. ABT-737 (1 μM) plus cycloheximide (CHX, 10 μg/mL) treatment for 6 h was used as a control. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4).

(D) Immunoblot of WT and Bax^−/−Bak^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 12, 24, or 48 h (n = 3).

Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.05 (^∗), p ≤ 0.01 (^∗∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figure S3.