Essential version 18

HepG2 cells were seeded on 15 µ-8 well glass bottom slide (Ibidi, Martinsried, Germany) and let grown overnight before transfection. Cells were transfected with 800 ng of mHilpda_mCherry plasmid complexed to polyethylenimine (PEI) (Polyscience Inc., PA, USA) in serum-free DMEM. After 6 h, the medium was replaced by complete medium. Next day cells were starved for 1h with HBSS 0.2% FA-free BSA. Medium was then replaced with QBT fatty acid uptake assay kit and after 4h incubation cells were imaged on a Leica TCS SP8 X system equipped with a 63x 1.20 NA water-immersion objective lens. Images were acquired sequentially using 512 x 512 pixels, and a total of 491 frames were acquired with a frame interval of ± 5 seconds. All images were deconvolved using Deconvolution Express modus with Huygens Essential version 18.10 (Scientific Volume Imaging, The Netherlands, http://svi.nl).
Further images were processed with Fiji to assign different coloring LUTs for visualization.

Cells derived from the parent strain JC-4066 were collected following the OCE process. Cells were resuspended in SC with 2% lactate; 0.05% Glucose and 2% glycerol (SCLGg) media and then treated with 2% GAL for 2 hours. Cells were then washed 2 times with PBS and placed on cover slips and imaged using a fully motorized Nikon Ti Eclipse inverted epi-fluorescence microscope. Z-stack images were acquired with 200 nm increments along the z plane, using a 60 X oil immersion 1.4 N.A. objective. Images were captured with a Hamamatsu Orca flash 4.0 v2 sCMOS 16-bit camera and the system was controlled by Nikon NIS-Element Imaging Software (Version 5.00). All images were deconvolved with Huygens Essential version 18.10 (Scientific Volume Imaging, The Netherlands, http://svi.nl), using the Classic Maximum Likelihood Estimation (CMLE) algorithm, with SNR:40 and 50 iterations. To measure the distance between the GFP and mCherry foci, the ImageJ plug-in Distance Analysis (DiAna) was used (Gilles et al. 2017) . Distance measurements represent the shortest distance between the brightest pixel in the mCherry channel and the GFP channel. Each cell was measured individually and > 50 cells were analyzed per condition per biological replicate.