Luperox

FCCP, oligomycin, rotenone, antimycin A, TBH70X, tert-Butyl hydroperoxide solution (Luperox), taxol, and Poly-l-Lysine (mol wt 70,000–150,000, 0.01%) were obtained from Sigma. Hoechst 33342, Mitotracker DeepRed, MitoSOX, and SYTOX Green were from ThermoFisher Scientific. TMRE was from Abcam. All compounds were stored at −20 °C except taxol and Hoechst (4 °C). Dyes were stored protected from light. FCCP, rotenone, antimycin A, Hoechst, MitoTracker DeepRed, and SYTOX were stored at −20 °C, TMRE was pre-diluted at 10 μM in media (10×) and aliquoted for single use. MitoSOX was likewise aliquoted for single use³⁰ .

Oligomycin, antimycin, FCCP, rotenone, AICAR, and Luperox (TBHP) were from Sigma. Antibodies against OXPHOS complexes (ab110413) and PGC-1alpha (ab106814) were from Abcam. Anti-GAPDH was from Cell Signaling (2118), anti-laminin from Dako (Z0097), anti-Tim23 (611223) and anti-RalA from BD (610221). Low glucose differentiation medium for immortalized human myoblasts was DMEM-F12 (US Biological) and for primary human skeletal muscle myoblasts (HSMM, Lonza) was DMEM (Gibco) supplemented with glucose (1 g/l) and 2% HS (Gibco). Galactose medium contained DMEM-F12 supplemented with galactose (1 g/l) and 2% HS.

Oxidative stress in both PMFs and HUAFs was induced by adding 10 μM reactive oxygen species (ROS) mimic tert-butyl hydroperoxide (tBHT; Luperox, 458139, Sigma Aldrich, St. Louis, MO, USA) to the culture media for 24 h. To serum starve the cells, DMEM with 1% PenStrep, supplemented with 1% FCSi for PMFs or 3% FCSi for HUAFs, was added to the cells. Different incubation times were tested for serum starvation (Figure S5). An incubation time of 24 h was determined as the most optimal time point. To induce hypoxia, cells in normal culture media were kept in a humidified incubator at 37°C under 1% O₂ for 24 h. Absolute Ct values of AF357425 and U6 expression are shown in Figure S6.

To determine ROS sensitivity in vitro, SD and SC (0.17% YNB, 0.5% ammonium sulfate, 1% complete SC medium without ammonium sulfate [Biomol, Germany]) media were solidified with 2% purified Bacto agar (BD) and the indicated concentrations of tert-butyl hydroperoxide (Luperox; Sigma-Aldrich) added. C. albicans cultures grown overnight were set to an OD₆₀₀ of 1 in sterile dH₂O, 10-fold serial dilutions were prepared (OD of 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵), and 5 μL from each dilution was spotted. The plates were subsequently incubated for 2 days at 37°C, and images were taken with a ProtoCol2 colony counter (Synbiosis, UK). The experiments were performed in biological triplicates.