Fig 1: Inhibitory effects of elaidic acid on autophagy induction by saturated FAs. (A, B) U2OS cells stably expressing GFP-LC3 were treated with 500 μM FA, or 10 μM of the autophagy-inducer subset from the ENZO SCREEN-WELL autophagy library, in the presence or absence of 500 μM EL for 6 h. Autophagy was assessed after fixation by measuring the area of LC3 dots within each cell. The relative difference with and without EL co-treatment was calculated and is reported as barchart in A. Representative images are shown in B. Scale bar equals 10 μm. (C) U2OS cells were treated with either vector (ethanol 0.5%), 500 μM OL, PA, or stearate in the presence or absence of 500 μM elaidate for 6 h. Thereafter, LC3 lipidation and p62 degradation and p38 activation were measured by immunoblot as indicators for autophagy. (D) U2OS cells treated as in C with OL, PA, and stearate in the presence or absence of elaidate, were harvested and analyzed with GC/MS to measure the intracellular FA concentration and to test the effect of EL on FA uptake into cells. Data are means ± SEM of at least three independent experiments (*=p < 0.05, **=p < 0.01, ***=p < 0.001, FA concentration in cells treated with a single FA compared to cells treated with vector control; #=p < 0.05, FA concentration in cells receiving EL co-treatment versus cells treated with single FA). (E, F) FYVE-RFP (E) and GFP-TFEB (F) stable expressing U2OS cells were treated as in C. After fixation, images were acquired and the area of FYVE+ dots and TFEB nuclear translocation were analyzed. Data are means ± SEM of at least three independent experiments (* = p <0.05, **=p < 0.01, ***=p < 0.001, FYVE dots surface or GFP-TFEBnuc/cyto ratio in cells with EL as compared to cells that were not co-treated with EL).