Mitochondrial toxicity assays

Multiplexed cytotoxicity assay — Human primary hepatocytes were grown on collagen I coated optical plates, cultured in hepatocytes culture media in a humidified 5% CO₂ atmosphere at 37 °C. Cells were incubated in the presence of MBX-4132 at 10 concentrations starting a 100 µM and serially diluted 3.16-fold for 24 h at 37 °C, incubated for 30 min with multiplexed fluorescent dyes (Hoescht, 6-carboxy-2’,7’-dichlorodihydrofluorescein diazetat, and TMRE) and imaged to allow visualization of nuclei, reactive oxygen species generation, and mitochondrial oxidation. A >3.5-fold induction of ROS was considered consistent with formation of ROS and a >2-fold change in TMRE signal indicated an increase or decrease in mitochondrial membrane potential⁵⁸.

Mitochondrial toxicity assay — HepG2 cells (ATCC) were seeded and cultured in media containing either glucose or galactose overnight. Test compounds were added at 8 concentrations (threefold serial dilution from 100 µM to 30 nM) and incubated with the cells for 24 h. Cell viability was measured by the alamarBlue method. The data in Supplementary Table ⁷ are inconsistent with MBX-4132 disrupting mitochondrial metabolic processes⁵⁹. Assays were performed at Eurofins Cerep Panlabs.