Fig. 3: GERVs are functional and accommodate the synthesis of neutral lipids.

a Left: Confocal microscopy of a Giant ER Vesicle (GERV) supplied with Oleoyl-CoenzymeA (OCoA) supplemented with NBD-Oleoyl-Coenzyme A (NBD-OCoA) and diacylglycerol (DAG) at 37 °C for 1 hour. Fluorescence signals at the bottom of the snapshots correspond to the profile indicated by the dotted line on the confocal images. Scale bar: 5 µm. Right: Plot showing the increase in NBD-OCoA fluorescence signal in the GERV membrane during the OCoA supply experiment. For both conditions, the number of independent experiments is N = 2, and the mean for each replicate is shown. A nested t-test was applied with a two-tailed P-value. P-values are shown. b Left: Confocal images of both KDEL-based Giant ER Vesicle (GERV) and GPI-based Giant Plasma Membrane Vesicle (GPMV) after 1 hour of OCoA supply. Fluorescence signals at the right corner correspond to the profile indicated by the dotted line on the confocal images. Scale bars: 5 µm. c Plot displaying the NBD-OCoA fluorescence signal in Giant ER Vesicles (GERVs) and Giant Plasma Membrane Vesicles (GPMVs) after 1 hour of feeding. For both conditions, the number of independent experiments is N = 2, and the mean for each replicate is shown. A nested t-test was applied with a two-tailed P-value. P-values are shown. Refer to Statistical Tests section or Source Data for statistical analysis and data sets. d Giant ER Vesicles were isolated from COS-7 cells overexpressing the DGAT1-EGFP and Sec61ß-mCherry. e Left: Isolated Giant Organelle Vesicles were subjected to the feeding conditions as in a. LipidTox was added to reveal membrane hydrophobicity. Fluorescence signals at the bottom of the images correspond to the profile indicated by the dotted line on the confocal images. Scale bars: 5 µm. Right: Box plot showing the LipidTox fluorescence in vesicles positive (+) or negative (--) for DGAT1. The number of independent experiments is N = 2 and a nested t-test was applied (f). Thin layer chromatography snapshot revealed by UV light indicates TAG-NBD synthesis in GERVs after 1 hour of feeding. Left: Commercial sample of OCoA-NBA; middle: Lipid extract from GERVs fed with OCoA-NBD and DAG; right: Commercial sample of TAG-NBD.