Lipid droplet, uptake, and lipid ROS assays

Lipid droplet content was assessed by staining with standard dye BODIPY 493/503 (Thermo Fisher). 27HCS or 27HCR cells (3000–10000 per well) were seeded in 96-well plates in regular media and grown to 60% confluence followed by BODIPY 493/503 staining following published methods⁸⁶. For the lipid uptake assay, BODIPY FL C16 (Thermo Fisher) was diluted to 5 mM in DMSO as stock solution. 27HCS or 27HCR cells were plated as above. The next day, cells were washed with cold PBS and BODIPY staining solution (2.5–100 µM BODIPY, 5 mM Fatty acid-free BSA in PBS) were added. Cells were then incubated at 37 °C for 25 min. Cells were washed three times with cold PBS and incubated with 4% paraformaldehyde (PFA) for 30 min at RT. Cells were further stained with 1 μg/ml Hoechst 33342 for 30 min at RT. BODIPY and nuclei Hoechst fluorescence were measured by a plate reader (TECAN Spark). For lipid peroxidation analysis, 5000 cells were plated per well in a 96-well plate and cultured for 48 h. Cells were then treated with 0.1% DMSO (vehicle), RSL3 (1 µM), and ML210 (5 µM) for 4 h. The treatment media was removed, and cells were washed once with HBSS, followed by labeling in 100 μl HBSS per well containing 5 uM BODIPY 581/591 C11 (Thermo Fisher) and 5 mM fatty acid-free BSA for 30 min at 37 °C. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at RT. Cells were further stained with 1 ug/ml Hoechst 33342 for 30 min at RT. BODIPY and nuclei Hoechst 33342 fluorescence were measured using a plate reader (TECAN Spark).