Fig. 3: Involvement of TNF-signaling in MVA-induced macrophage cell death.

a, b D7 differentiated wt (black bars) or Bax/Bak-DKO (gray bars) Hoxb8 macrophages were infected with MVA at a MOI of 2 and harvested after 22 h by accutase treatment followed by annexin/PI staining (a) or active caspase-3 staining (b) and flow cytometry analysis. Inhibitors were used at the following concentrations where indicated: QVD, 20 µM; Nec-1, 10 µM. c Supernatants of d7 differentiated wt (black bars) or Bax/Bak-DKO (gray bars) Hoxb8 macrophages infected with MVA at an MOI of 2 for 22 h were analysed for secreted TNF by ELISA. d D7 differentiated wt (black bars) or Bax/Bak-DKO (gray bars) Hoxb8 macrophages were infected with MVA at a MOI of 2 and harvested at the indicated time points by accutase treatment. Where indicated, samples were concomitantly treated with anti-TNF antibody (1:1000). Cells were analysed for cell death by annexin V/PI staining followed by flow cytometry. The rate of dead cells was determined as a percentage of annexin/PI-positive cells (cells positive for annexin V or PI or both). e D7 differentiated wt or TNF^−/− Hoxb8 macrophages were infected with MVA at an MOI of 2 and harvested at the indicated time points by accutase treatment. Cells were lysed in Laemmli sample buffer and samples were boiled at 95 °C for 5 min. Samples were subjected to SDS-PAGE on 4–20% TRIS-glycine gels, transferred onto nitrocellulose membranes, and probed with the antibodies indicated. The pan-caspase inhibitor QVD was used at a concentration of 20 µM where indicated. GAPDH served as a loading control. Blots are representative of at least two independent experiments. Shown are means/SEM of n = 4 (a, b, d) or n = 6 (c) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns nonsignificant (p ≥ 0.05).