Cholesterol Extraction and Measurement in Cells

SKMEL2 NT or GRAMD1B shRNA cells (5 × 10⁶-10 × 10⁶) were trypsinized, counted, and centrifuged to obtain cell pellets for cholesterol extraction. Half of the cells were saved for protein extraction and quantification. The remaining half of the cells were washed 1× in PBS and then extracted with 200 μL of chloroform:isopropanol:NP-40 (7:11:0.1). Extracts were sonicated at an amplitude of 10%, with 10 total pulses of 1 second. Extracts were centrifuged for 10 minutes at 15,000 × g, and the organic phase was transferred to a new tube. Tubes were air dried at 55°C to remove chloroform. The samples were then dried in a speed-vac to remove the remaining organic solvent for 20 minutes. Dried lipids were resuspended with 200 μL of Cholesterol Assay Buffer by vortexing until homogeneous. A standard curve was generated using Cholesterol standards in the kit. Cholesterol levels were quantified using the fluorescent Abcam cholesterol/cholesteryl kit as per manual instructions. For experiments in Fig. 5H and ​andII and Fig. 6A, we used Promega's cholesterol/cholesterol ester glo assay kit (J3190) as this was more convenient in a 96-well format and amenable to high-throughput analysis of intracellular cholesterol. For these experiments, 8,000 NT or KO cells were seeded in a 96-well plate with at least 6 wells per condition. The next day, 3 wells per condition were used to measure cholesterol as per the manual instructions. The remaining 3 wells were used to quantify the number of viable cells using Promega's CellTiter-Glo kit (G7570). Cholesterol luminescence values were normalized to cell viability and then a standard curve was used to obtain cholesterol concentration per sample (μmol/L).