Metabolic Stress Response in Mitochondrial Dysfunction

MELAS cybrid cells and Rieske KO fibroblasts were seeded in 2.5 mM glucose with 22.5 mM galactose media at a density of 1.0×10⁵ cells per well in 6-well plates. ND1 and ND6 mutant cybrid cells were seeded in 25 mM galactose media with 0 mM glucose at a density of 1.0×10⁵ cells per well in 6-well plates. Stocks of doxycycline hyclate (Sigma, D9891), pentamidine isethionate (Sigma, 1504900), and retapamulin (Sigma, CDS023386) were dissolved in DMSO at 100 mM. Compounds were added to wells at the time of seeding at a final concentration indicated in the figures and figure legends (1 nM-100 μM). The p38 inhibitor, SB203580 (Cell Signaling, 5633S) was dissolved in DMSO and used at a final concentration of 25 μM as previously described ^{33 (link)}. MELAS and Rieske KO cells were counted after 48 hours incubation, unless otherwise noted. ND1 and ND6 were maintained for the time point indicated in the figure legends with galactose (0 mM glucose) media. For long term survival experiments in low-glucose (e.g. MELAS/Rieske cells) or galactose (e.g. ND1/ND6 cells) media, fresh media and drug were replaced every 24 or 48 hours.