Image analysis workflows for LD and ER quantifications. (A) Cells stained with BODIPY 493/503 (LDs) and Hoechst (nuclei) were imaged on multiple z slices using a confocal microscope. Z-stack projection (max intensity) images were generated using Fiji and processed using CellProfiler. Cells were manually segmented and LDs identified as objects ≥2 pixels of diameter. Multi-parametric object measurements were performed on the identified LDs. (B) Cells were treated with OA at 50 µM for 6 h before imaging. Three channels were acquired: LDs (stained with HCS DeepRed LipidTox), the ER (stained with the ER marker mScarlet-ER), and MOSPD2/VAP-A (tagged with GFP). Cells were manually segmented and masks of the cytoplasm (i.e., without the nuclei) were generated with CellProfiler. LDs were identified as objects ≥4 pixels of diameter. Multiple areas (2-pixel wide) from 0 to 20 pixels around each LD were added. Multi-parametric measurements were performed for each area around LDs in the red (mScarlet-ER) and green (GFP-MOSPD2/VAP) channels.