Ant a

TLR-2 inhibitor (CUCPT-22, 1 μM), ER-stress inhibitor (4-phenyl butyric acid, 4-PBA, 10 µM), mtROS inhibitor (YCG063, 10 µM), mtROS inducer (antimycin A, Ant A, 50 µM), TNF-α biosynthesis inhibitor pentoxifylline (Pentox, 1 mM), and mt-Ca²⁺ uniporter blocker (Ru360, 10 µM) were purchased from Sigma. Caspase-8 inhibitor (Z-IETD-FMK, 10 µM) was purchased from Biovision. (Ca²⁺)_c monitoring dye (Fluo-3/AM, 2 µM) and mt-Ca²⁺ monitoring dye (Rhod-2/AM, 5 µM) were purchased from Invitrogen. Mitochondrial superoxide indicator (Mitosox, 5 μM) was purchased from Molecular Probes. HKM were pretreated with specific inhibitors for 1 h prior to infection with M. fortuitum. The doses of different inhibitors were selected on the basis of inhibitor specificity and cytotoxicity. The HKM treated with the indicated concentrations of the inhibitors remained as viable as control HKM at all-time points as determined by the trypan blue (0.4%) dye exclusion method and were maintained during the entire course of the experiment (16 (link), 36 (link), 37 (link)).

Mitochondrial respiration was assessed in non-differentiated and differentiated SH-SY5Y cells and in primary cortical neurons treated as described above. Wherever indicated, cells were treated for 30 min with 3 μM xestospongin C (XeC; Tocris Cat. no. 1280). OCR was measured using the Seahorse® XF24 or XF96 analyzers (Agilent) as an indication of mitochondrial respiration. The analysis was performed in base DMEM (Sigma-Aldrich, Cat no. 5030) supplemented with 25 mM glucose, 10 mM glucose, or 10 mM pyruvate (pH 7.4) as substrate for SH-SY5Y cells; or in base DMEM supplemented with 10 mM glucose and 0.223 mM pyruvate for primary neurons (pH 7.4). The OCR was measured at baseline and followed by sequential stimulation with 1 μM oligomycin A (Sigma-Aldrich, Cat no. 75351), 1.5 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma-Aldrich, Cat no. C2920), and 0.5 μM Ant A (Sigma-Aldrich, Cat no. A8674) plus 0.5 μM Rot (Sigma-Aldrich, Cat no. R8875). Basal respiration, maximal respiration, and oligomycin-sensitive respiration were calculated using the Seahorse XF Cell Mito Stress test report generator from Wave 2.6.1 software (Agilent). OCR measured in pmol O₂/min was normalized to cell number or protein content.