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. 2020 Dec 7;13(1):e13207. doi: 10.15252/emmm.202013207

Figure 1. Identification of the putative Cyt‐bd inhibitor ND‐011992.

Figure 1

  1. Molecular structure of ND‐011992.
  2. Effect of ND‐011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND‐011992 alone (brown square), and a dose range of ND‐011992 in the presence of Q203 (red squares) for 15 h before quantification of intracellular ATP levels.
  3. Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor.
  4. Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyzer. 12 μM ND‐011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalized to the last basal OCR reading before drug injection. OCR: oxygen consumption rate. The dotted lines indicate the timepoints of drug injection.
  5. Dose‐dependent inhibition of M. bovis BCG OCR by ND‐011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyzer platform. For each condition, OCR readings were normalized to the last basal OCR reading before drug injection.
  6. Effect of ND‐011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMV OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.

Data information: Data are expressed as the mean ± SD for each condition of a representative experiment. Experiments in (B) and (E) were performed with 2 technical replicates and independently repeated at least twice. In (D) and (F), each experiment was performed with 3 technical replicates and independently repeated at least once.