Nunclon δ surface plates

Cytosolic extraction using digitonin was performed as previously described^{59 (link)} with the exception that digitonin was used at 25 μg/ml. Briefly, cells were seeded in six-well Nunclon Δ Surface plates (Nunc, 150,000 cells per well) in DMEM, 10% FCS (Gibco/Sigma) and next day treated with free fatty acids as indicated. Cells were harvested and counted, with 175,000 cells pelleted and extracted with 300 μl of 25 μg/ml Digitonin (300410, Calbiochem) in subcellular fractionation buffer (HEPES-KOH 20mM, sucrose 250 mM, KCl 10 mM, MgCl₂ 1.5 mM, EDTA 1 mM, EGTA 1 mM, dithiothreitol 8 mM, Pefabloc 1 mM (Fluka), at pH 7.5), a concentration that had been determined to be optimal (concentrations ranging from 12.5-50 μg/ml were tested and 25 μg/ml showed good cytosolic extraction, with minimal lysosomal damage). Cells were incubated for 10 min on ice with intermittent vortexing. The samples were then spun down (90 s, 13,000 r.p.m., 4 °C) and the supernatant quickly transferred to a new tube to be used in downstream cathepsin activity assays. For each condition 175,000 cells were also extracted in 0.1% TritonX-100 as above to obtain total cell lysates for assessing total cathepsin activity.