BODIPY 493/503 staining of lipid droplets

HepG2 cells were grown in glass-bottom dishes and treated as mentioned above. They were then incubated for 10 min in the dark with 0.2 μM boron-dipyrromethene (BODIPY) 493/503 (D3922, Thermo Fisher Scientific, MA, USA), which labels specifically intracellular neutral lipids, and 1 min with 1 μM DAPI (10236276001 Roche, Switzerland) to stain the nuclei. Imaging of the cells was performed on a Zeiss LSM 870 confocal microscope. The channels' exposure times were independently set to maximize the signal while minimizing the number of cells with expression levels above saturation to optimize the assay's dynamic range. Once set for each channel, all images in that channel were collected at the same exposure. We used ImageJ software (version 1.8.0, NIH, USA) to analyze the images. The intracellular lipid accumulation was quantified by dividing the BODIPY fluorescence intensity over that of the DAPI. For each treatment condition (untreated, steatotic, and Ex-4-treated steatotic cells), two independent researchers analyzed 200 individual cells from three different experiments.