Fig. 5

Mtb triggers p38 MAPK-dependent necrosis by opening of the mitochondrial permeability transition pore (mPTP). a Protective effect of cyclosporine A (CsA; 10 µM) in Mtb- infected MRC-5 lung fibroblasts (MOI 10). Viability was quantified 72 h after infection using Prestoblue. b Fluorescence microscopy of uninfected and infected (MOI 5) J774.2 Mφ stained with tetramethylrhodamine (TMRM) 48 h post infection (scale bar: 100 µm). Images are representative of two independent experiments. c Quantification of cytosolic and mitochondrial CypD following Mtb infection. MRC-5 lung fibroblasts were infected with Mtb (MOI 10), treated with dexamethasone (5 µM), doramapimod (10 µM), or CsA (10 µM) and lysed for the isolation of mitochondria 5 h post infection. Equal amounts of protein from the mitochondrial and cytosolic fractions were subjected to western blot analysis and the levels of CypD were measured. Voltage-dependent anion-selective channel 1 (VDAC-1) was used as a loading control. d Expression of hexokinase II (HKII) in Mtb- infected MRC-5 lung fibroblasts. Lysates were obtained 24 h after infection and were analyzed by qRT-PCR. e Quantificiation of mitochondrial HKII following Mtb infection. J774.2 Mφ were infected with Mtb, treated with dexamethasone (5 µM) or doramapimod (10 µM), and mitochondria were isolated 5 and 24 h post infection. Equal amounts of protein were subjected to western blot analysis and the levels of HKII were measured. VDAC-1 was used as a loading control. f Quantification of HKII in the cytosol of MRC-5 lung fibroblasts. Whole-cell lysates were obtained from infected untreated cells and from infected cells treated with doramapimod (10 µM) or BMS-582949 (10 µM) 48 h post infection. HKII was quantified by western blot analysis using β-actin as a loading control. Images are representative of two individual experiments. Representative data from at least two experiments with multiple replicates are shown in a and d. Results are expressed as mean ± SEM and experiments were analyzed using unpaired t-test (ns not significant; **p ≤ 0.01; ***p ≤ 0.001)