Figure 1.

IFNγ primes macrophages for TLR-induced cell death

(A and B) Wild-type (WT) bone-marrow-derived macrophages (BMDMs) were treated with (A) IFNγ (50 ng/mL) or (B) IFNβ (1,000 U/mL, n = 5) overnight, then with either LPS (50 ng/mL, n = 5), Pam3CSK4 (P3C, 500 ng/mL, n = 6), or PolyI:C (10 μg/mL, n = 6) for 24 h. Cell death was assessed by propidium iodide (PI) exclusion as measured by flow cytometry.

(C) WT or Tnf^−/−Fasl^gld/gldTrail^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) (left), or P3C (500 ng/mL) (right) for 24 or 48 h ± TNF (100 ng/mL). Cell death was assessed by PI exclusion as measured by flow cytometry (n = 5).

(D) Immunoblot of WT BMDMs treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 8, 12, or 24 h. Treatment with the BCL-2, BCL-XL, and BCL-W inhibitor, ABT-737 (737, 1 μM) and cycloheximide (CHX, 10 μg/mL) ± Q-VD-OPh (QVD, 20 μM) for 4 h was used as a control (n = 3).

(E) Immunoblot of Tnf^−/−Fasl^gld/gldTrail^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 12, 24, or 48 h (n = 2).

Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figures S1 and S2 and Videos S1, S2, S3, and S4.