at 37 °C in DMEM supplemented with 10% FBS, 50 μg/ml uridine , 1% MEM non-essential amino acids , 10 μM palmitate conjugated to 1.7 μM bovine serum albumin , and 0.001% DMSO . Cells were passaged approximately every 5 days . Oligo-treated healthy control cells were cultured in the same media as control cells supplemented with 1 nM Oligomycin starting on Day 15.
Brightfield microscopy images were taken before each passage using an inverted phase-contrast microscope . Cell counts, volume, and death were determined at each passage using the Countess II Automated Cell Counter . Growth rates were used to determine replating density, by pre-calculating the number of cells needed to reach ~90% confluency at time of the next passage. Cells were never plated below 200,000 cells or above 2.
The final Seahorse injection included Hoechst nuclear fluorescent stain to allow for automatic cell counting. After each run, cell nuclei were counted automatically using the Cytation1 Cell Imager , and raw bioenergetic measurements were normalized to relative cell counts on a per-well basis. ATP metrics were determined using the P/O ratios of OxPhos and glycolysis as previously described by Mookerjee et al.
To assess if increased ECAR in experimental conditions was due to non-glycolytic activity, a glucose-dependency test was performed using the Seahorse XF Glycolysis Stress Test Kit . Prior to extracellular flux measurements, young healthy control and young SURF1 cells were grown overnight in differing nutrient conditions: physiological 5.5 mM glucose, 0 mM glucose, 25 mM glucose. The glycolysis stress test kit was performed according to the manufacturer’s protocol.
NGS Libraries were generated using an enzymatic DNA fragmentation approach using Ion Xpress Plus Fragment Library Kit. Libraries were diluted at 100 pM before sequencing and pooled by a maximum of 25 samples. Sequencing was performed using an Ion Torrent S5XL platform using Ion 540 chipTM. Signal processing and base calling were done by the pre-processing embedded pipeline.