Figure 4

PFT-mediated caspase activity simultaneous to necroptosis promotes alarmin release and inflammation. (a) Western blots for S100A9 and HSP60 from A549 cells challenged with rPly after pre-treatment with Z-VAD-FMK (Zvad, 100 μM), or necrosulfonamide (NSA, 10 μM). Blots images were cropped from separate gels, uninfected control (first lane) vs rPly challenged cells (2–4 lanes) are shown side-by-side in the same gel. Densitometry from 3 separate gels is shown next to the immunoblots. (b) IL-6 (pg/ml) in supernatants of THP-1 macrophages challenged with the supernatants of A549 cells that were challenged with rPly (subsequently blocked by antibody) pre-treated with Z-VAD-FMK (Zvad, 100 μM), or mock treated. (c) Western blot for pMLKL, cleaved caspase-2, and cleaved caspase-4 and actin (loading control) in the lungs of mice (n = 3–4) infected Sma via intratracheal route vs uninfected control. Blots images were cropped from separate gels, uninfected control (first lane) vs Sma infected mice (2–5 lanes) are shown side-by-side in the same gel. Densitometry from 3 separate gels is shown next to the immunoblots. Extra mock infected controls for densitometry were ran in separate gel. (d) Healthy baboons were challenged intrabronchialy with Spn using a video assisted bronchoscope and sacrificed 5–7 days after infection for tissue collection. Western blot for pMLKL, cleaved caspase-2, cleaved caspase-4 cleaved caspase-10 and actin (loading control) in the lungs of Spn infected baboon’s vs uninfected control. Blots images were cropped from separate gels, uninfected control (first lane) vs Spn infected baboon’s (2–4 lanes) are shown side-by-side in the same gel. For multiple group comparisons Kruskal-Wallis test with Dunn’s multiple-comparison post-test was used: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. For in vitro experiments averaged data from >3 separate experiments are shown.