C9754

A stock solution of colchicine (4 mg/ml in sterile ddH₂O, C9754, Sigma-Aldrich) was prepared and stored at −20 °C until the day of treatment. Immediately prior to administration, the colchicine stock solution was diluted to 0.1 mg/mL in sterile ddH₂O water. An equal volume of colchicine (0.4 mg/kg) or vehicle was administered to mice (6 weeks old) daily via I.P. injection for 7 days. Muscle tissues were harvested from treated mice on the day after the final treatment.

A subset of animals was treated with either colchicine (0.4 mg/kg/day; Col; C9754, Sigma) or 0.9% saline as vehicle (Veh) via intraperitoneal injection for 3 days prior to tissue collection as previously described [21 , 40 ]. Constituents of the autophagosome were measured via immunoblotting in both mitochondrial fractions and whole muscle lysates to accurately capture mitophagy and autophagy. To calculate flux, the Veh values were subtracted from the mean Col value for the corresponding timepoint.

For Col (C9754, Sigma) treatment, Col was dissolved in PBS and injected intraperitoneally in mice at 18, 6, and 2 h before heart harvest at doses of 4, 1, and 1 µg/g body weight, respectively. For CQ treatment, CQ was dissolved in PBS and injected intraperitoneally in mice at 48, 24, and 2 h. before heart harvest at doses of 30, 30, and 50 µg/g body weight, respectively. After the treatments above, mice were euthanized, hearts were immediately removed and rinsed in ice-cold PBS, and then frozen in liquid nitrogen.

Experiments with isolated fibers were performed the day after isolation. For prolonged (15 hr) nocodazole treatment, nocodazole (M1404, Merck) was added for a final concentration of 4 μg/ml at this step. When palmitic acid treatment is indicated, this was added for a final concentration of 0.5 mM at this step as well. Palmitic acid was dissolved to a 200 mM solution in 1:1 ethanol and α-minimal essential medium (MEM), from which a 16× solution containing 100 mg/ml fatty acid-free bovine serum albumin (BSA) was made. Non-treated fibers were treated with BSA without palmitic acid. When indicated C2 ceramide (50 μM) (860502, Avanti), Paclitaxel (10 μM) (T7402, Merck), kinesore (6664, Tocris), or colchicine (25 μM) (C9754, Sigma) was added 2 hr prior to imaging/lysing whereas nocodazole (13 μM) was added 4 hr prior unless otherwise mentioned. For signaling analyses, 30 nM insulin (Actrapid, Novo Nordisk A/S) was added 15 min prior to lysing, for microscopic analyses 30 nM insulin was added 15–30 min prior to imaging. For fixation fibers were incubated with 4% paraformaldehyde (Electron Microscopy Sciences) in phosphate-buffered saline (PBS) for 20 min.