Journal: Frontiers in Immunology

Article Title: Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production

doi: 10.3389/fimmu.2021.742370

Figure Lengend Snippet: 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.

Article Snippet: PF-573-228 and necrostatin-1 were purchased from MedChem Express (Monmouth, NJ), while necrostatin-1s was purchased from New England Biolabs (Ipswich, MA).

Techniques: Infection, Western Blot, Negative Control, Activation Assay, Positive Control, Control, Staining, Flow Cytometry, CyQUANT Assay, Glo Assay

Journal: Frontiers in Immunology

Article Title: Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production

doi: 10.3389/fimmu.2021.742370

Figure Lengend Snippet: Expression of FAK controls Mtb survival. (A) Mtb was treated with the indicated concentrations of FAK inhibitors (in the absence of macrophages), PF-573-228 and PF-562-271, for 5 days, after which Mtb growth was assessed using the resazurin viability assay. Relative viability was normalized to 100% using untreated controls. (B) THP-1, THP-FAKi, and THP-FAK + macrophages, or (C) RAW 264.7 and RAW 264.7-FAKi macrophages were infected with Mtb-luciferase at an MOI of 10 for 6 days. Infected macrophages were lysed at the indicated time points post-infection and the resultant luminescence signal (RLU) was measured as a proxy for the relative number of viable of Mtb. (D, E) THP-1 macrophages were mock treated or pre-treated with (D) 30 µM necrostatin-1 or (E) 5 µM GSK-872 for 24 h, and subsequently infected as in (B) . Infected macrophages were lysed at the indicated time points post-infection, and the resultant luminescence signal (RLU) was measured as a proxy for the relative number of viable of Mtb. Error bars represent the mean ± SD of three independent biological replicates. ** p < 0.01, *** p < 0.001.

Article Snippet: PF-573-228 and necrostatin-1 were purchased from MedChem Express (Monmouth, NJ), while necrostatin-1s was purchased from New England Biolabs (Ipswich, MA).

Techniques: Expressing, Viability Assay, Infection, Luciferase

Journal: Frontiers in Immunology

Article Title: Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production

doi: 10.3389/fimmu.2021.742370

Figure Lengend Snippet: Expression of FAK induces production of ROS to control Mtb infection. (A) THP-1, THP-FAKi, and THP-FAK + macrophages were infected with Mtb-tdTomato (red signal) for 6 days, after which cells were stained with DCFH-DA (5 μM, green signal) for 30 minutes at 37°C. Representative Bright Field and fluorescence images of uninfected and Mtb-infected (red) macrophages (green, ROS), are shown using a 20x objective. Scale bar, 100 μm. (B) THP-1 macrophages were infected and stained with DCFH-DA as in (A) and the amount of ROS production was quantified at the indicated time points post-infection using a fluorescence plate reader at 485 nm/535 nm. (C) RAW 264.7 macrophages were infected and stained as in (A) and the amount of ROS production was quantified at day 6 post-infection. (D) THP-1 and THP-FAK + macrophage were pre-treated with 10 mM N-acetyl-cysteine (NAC) or 25 µM GSK2795039 for 24 h and then infected with Mtb-luciferase at an MOI of 10. Infected macrophages were lysed at day 6 post-infection and the resultant luminescence signal was measured as a proxy for the relative number of viable of Mtb. RLU signals were normalised to mock treated THP-1 cells as 100% and depicted as % Mtb survival. (E) THP-1, THP-FAKi, and THP-FAK + macrophages were mock treated or pre-treated with 30 µM necrostatin-1 for 24 h, after which cells were infected with Mtb at MOI of 10. At indicated time points post-infection, infected macrophages were stained with DCFH-DA as in (A) and fluorescence signals for ROS production was measured. Error bars represent the mean ± SD of three independent biological replicates. * p < 0.05, *** p < 0.001.

Article Snippet: PF-573-228 and necrostatin-1 were purchased from MedChem Express (Monmouth, NJ), while necrostatin-1s was purchased from New England Biolabs (Ipswich, MA).

Techniques: Expressing, Control, Infection, Staining, Fluorescence, Luciferase