Fig. 3

Mitophagy is regulated by BNIP3 in Mtb-infected macrophages. A RAW 264.7 cells were infected with Mtb at an MOI of 1. Western blot analysis was performed using mitochondrial and cytosolic fractions from Mtb-infected macrophages. B RAW 264.7 cells were pretreated with FM19G11 (20 μM) or NAC (20 mM) for 1 h and then infected with Mtb (MOI = 1) for 3 h. Cell lysates were collected 48 h post infection. C Mitophagy was measured in RAW 264.7 cells using a mitophagy detection kit (n = 5 per group). D The fluorescence intensity ratios of mitochondrial DNA in figure S3. The data are presented from at least three independent experiments (means ± SD of n = 8). E BNIP3-knockout (BNIP3-KO) RAW 264.7 cells were separated into cytosolic and mitochondrial fractions after Mtb infection for 48 h (MOI = 1). RAW 264.7 cells were treated with CoCl₂ (200 μM) for 24 h. Protein levels of LC3 and BNIP3 were measured by western blotting. COX IV and β-tubulin were used as mitochondrial and cytosolic fraction loading controls, respectively. F Mitochondrial membrane potential (MMP) was measured in RAW 264.7 cells using JC-1 staining. Fluorescent intensity of JC-1 was detected at excitation wavelength 488 nm and emission wavelength 530 nm using flow cytometry (n = 4 per group). G Mtb-infected wild-type (WT) and BNIP3-KO RAW 264.7 cells were incubated with MitoSOX Red to measure mitochondrial ROS (mROS) levels. The mROS levels were analyzed using flow cytometry (n = 4 per group). H The fluorescence intensity ratios of Tom20 and Tim23 in figure S4. The data are presented from at least three independent experiments (means ± SD of n = 5). I Oxygen consumption rate (OCR) was measured after sequential treatment with oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone (n = 5 per group). The data are presented as means ± SD of at least three independent experiments; *p < 0.05, **p < 0.01