Fig. 4: Impaired fatty acid metabolism in ESYT1/2-VAPB-deficient cells.

a Representative western blot showing ESYT1, ESYT2 and VAPB protein in HeLa cells following CRISPR-Cas9 mediated knockout of ESYT1, ESYT2, VAPB and ESYT1 and ESYT2. GAPDH, loading control. * non-specific immunoreactive band. b 3D rendering images of Control and KO cells stained for LDs with HCS LipidTox Deep Red Neutral Lipid Stain (various colors) and mitochondria with TOMM20 antibody (red). Prior to staining ESYT1/2/VAPB^KO and Control HeLa cells were treated with 400 μM fatty acids (oleic acid and palmitic acid; 2:1) for 4 h. Scale bars: 0.5 μm. c Quantification of volume occupied by LDs per cell from experiments shown in panel B. Control n = 35, ESYT1^KO n = 44, ESYT2^KO n = 42, VAPB^KD n = 34, DKO n = 35 cells. d Lipidomic analysis showing triacylglycerol content in HeLa cells treated with 400 μM oleic acid and palmitic acid (2:1) for 4 h. n = 6 biological replicates. e, f HeLa cells were pulsed for 6 h with radiolabeled fatty acid (¹⁴C oleic acid) conjugated to 1% BSA and chased for 4 h in low glucose medium without fatty acids (starvation medium). Data indicates rate of ¹⁴C- oxidation (LD-derived fatty acid oxidation) and ¹⁴C remaining in triglyceride following the chase period. Bar graphs represent the mean ± SEM from 3 (e) and 4 (f) biological replicate experiments. g BODIPY C₁₆ in LDs of cells that were pulsed with BODIPY C₁₆ for 6 h, washed, and incubated in starvation medium for 4 h. n = 6 (VAPB^KD, DKO) or 7 (Control, ESYT1^KO, ESYT2^KO) biological experiments. h Western blot showing steady state level of phosphorylated and total ATGL and HSL protein in cells. * denotes non-specific immunoreactive band. i Flux of ¹³C fatty acids into TCA cycle and non-mitochondria metabolites (n = 4 per group). The ¹³C-fatty acid mix contained myristic (0.2%), palmitoleic (9.4%), palmitic (38.9%), margaric (0.3%), linoleic (10.7%), oleic (26.9%), elaidic (1.6%), and stearic (1.6%) acid. For (c– i) bar graphs represent the mean ± SEM, *p < 0.05 vs. Control using one-way ANOVA with Bonferroni’s multiple comparison test. See also Supplementary Fig. 4 and Supplementary Data 3.