Fig 1. Autophagy and mitophagy were increased in PA-treated cells.

A. Primary human aortic endothelial cells (HAECs) were treated 0 or 0.3mM palmitic acid (PA) for 24 hours. Treated cells were stained with mitochondria marker Mito, autophagy marker LC3, and lysosome marker Lamp1. The numbers of mitochondria co-localized with LC3 or with Lamp1 per cell were quantified. Representative immunostaining images and quantification of 3 independent experiments demonstrated increased co-localization of mitochondria with LC3 and Lamp1 in PA treated cells. At least 25–30 cells per experiment were analyzed. B. HAECs were treated with 0 or 0.3mM PA for 24h and 5nM bafilomycin A1 during the last 6h prior to fixation. Representative electron microscopy photomicrographs showed the formation of autophagosomes containing heterogeneous cytoplasmic materials (black arrows) and mitophagosomes containing mitochondria fragments (white arrows). Asterisk indicated normal mitochondria. At least 10–15 cells per condition were imaged. Quantification showed the number of autophagosome and mitophagosome was increased by PA treatment. C. HAECs were treated with 0 to 0.5mM PA for 24h. Representative images of western blot and quantification from 3 independent experiments showed dual effects of PA on PINK1 and Parkin expression. */#P< 0.001 vs. PA (0mM). D. HAECs were treated with 0 or 0.3mM PA for 24h. Representative immunostaining images of 3 independent experiments demonstrated co-localization of PINK1 and Parkin with mitochondria in perinuclear area in PA treated cells.