Oxygen Consumption Assays.

Oxygen consumption in whole bacteria was measured using methylene blue or the MitoXpress Xtra–Oxygen Consumption Assay (Luxcel Biosciences).

Methylene blue-based assay. Mycobacteria culture adjusted to an OD₆₀₀ of 0.3 were preincubated for 4 h in 2-mL screw-cap tubes in the presence of Q203 at 400 nM, BDQ at 500 nM, or 1% DMSO (vehicle control). Methylene blue at 0.001% was added to each tube. The tubes were then tightly sealed, an incubated in an anaerobic jar to avoid oxygen leak.

MitoXpress-based assay. The assay was performed in black 96-well plates (flat, clear bottom). One hundred fifty microliters of mycobacteria culture adjusted to an OD₆₀₀ of 0.3 were preincubated for 6 h in the presence of Q203, BDQ, or 1% DMSO. Ten microliters of the MitoXpress oxygen probe was added to each well that was covered with a layer of high-sensitivity mineral oil to restrict oxygen back diffusion. Fluorescence (Ex: 380 nm, Em: 650 nm) was recorded on a BioTeK CYTATION 3 multimode reader.