Fig. 6: ESYT1, ESYT2 or VAPB depletion sensitizes cells to palmitic acid-induced lipotoxicity and cellular stress.

a Schematic representation of fatty acid trafficking at LD, mitochondria and ER contacts in the presence of protein-mediated fatty acid transfer (efficient fatty acid trafficking) and with inefficient fatty acid trafficking mediated by disruption to the ESYT/VAPB complex. Created in BioRender. Keenan, S. (2025) https://BioRender.com/n75s805. b–f Lipidomic analysis showing abundance of lipids associated with lipotoxicity including diacylglycerol, ceramide, sphingomyelin (SM), cardiolipin, and lysophosphatidylcholine (LPC). Bar graphs represent the mean ± SEM from 6 biological replicate experiments. *p < 0.05 vs. Control using one-way ANOVA with Bonferroni’s multiple comparison test. g Representative Western blot and quantitation of ER stress marker proteins. Bar graphs represent the mean ± SEM from n = 4 biological replicate experiments of BIP, ATF5, p-PERK (Thr980), PERK and PDI. ATF5_s/ATF6_FL indicates ratio of protein content of short and full length ATF6. GAPDH was used as loading control. Data analysed using one-way ANOVA and a Kruskal-Wallis post hoc test. p < 0.05 vs. Control. (h) Representative Western blot and quantitation of p-ERK (Thr202/Tyr204) and ERK, p-JNK (Thr183/Tyr185) and JNK. GAPDH was used as a loading control. Bar graphs represent the mean ± SEM. (p-JNK/JNK n = 4 and p-Erk1/2 / ERK1/2 n = 5 independent experiments). Data analysed using one-way ANOVA and a Kruskal-Wallis post hoc test. *p < 0.05 vs. Control. i Representative Western blot and quantitation of pyroptosis markers with palmitic acid-induced stress in control and KO cells. In all experiments in this panel, HeLa cells were treated with palmitate (500 µM) conjugated to 1% BSA for 4 h. Bar graphs represent the mean ± SEM protein content of HMGB1 (n = 6 for Control, ESYT2^KO, VAPB^KD and DKO, n = 5 ESYT1^KO), cleaved caspase 1 (n = 4), ratio of full length and splice variant of Gasdermin (n = 4) from independent experiments. GAPDH was used as loading control. Data analysed using one-way ANOVA and a Kruskal-Wallis post hoc test. *p < 0.05 vs. Control.