Figure 5.

FAK controls necroptotic cell death in macrophages during Mtb infection. (A) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, and cell lysates were prepared at the indicated days post-infection (d.p.i.). Total and cleaved caspase 3, and total FAK protein levels were analyzed by western blotting. Non-infected THP-1 cell lysates were used as a negative control for caspase 3 activation, while lysates containing cleaved caspase-3 (#9663, Cell Signaling Technologies) were used as a positive control. β-tubulin was used as loading control. (B) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected as in (A) and at the indicated days post-infection, cells were stained with annexin V and FVS780. Stained cells were analyzed by flow cytometry to quantify the percentage of healthy, apoptotic, and necrotic cells as described in the Methods. (C) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, and lactate dehydrogenase (LDH) released in culture supernatants were assessed using the CYQUANT LDH kit at indicated days post-infection. The amount of LDH in the supernatant is proportional to the measured absorbance at 490 nm. (D) THP-1, THP-FAKi and THP-FAK⁺ macrophages were mock treated or pre-treated with 1 µg/ml LPS for 4 h, followed by treatment with 5 µM nigericin for 24 h Macrophage viability was assessed using the Cell Titre-Glo assay. (E–G) THP-1, THP-FAKi, and/or THP-FAK⁺ macrophages were mock treated or pre-treated with (E) necrostatin-1s (Nec-1s, 10 µM), (F) GSK-872 (5 µM), or (G) necrosulfonamide (NSA, 10 µM) for 24 h Cells were then infected with Mtb at an MOI of 10 for 6 days, and cell viability was assessed using the Cell Titer-Glo assay. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, ***p < 0.001, ns, non-significant.