Extended Data Fig. 6: Leucine-mediated activation of mTORC1 inhibits autophagy in macrophages.
From: High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy
(a) Immunoblot analysis of phospho- and total ULK1 levels in macrophages incubated with amino acid-free medium with and without leucine for the indicated times. (b, c) The autophagy marker LC3 was evaluated in macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes using (b) immunoblot analysis or (c) quantification of LC3 intensity (+aa: n = 52; -aa: n = 52; Leu: n = 52 cells) and number of puncta (+aa: n = 97; -aa: n = 87; Leu: n = 97 cells) by immunofluorescence microscopy. (d) Phospho- and total ULK1 levels were determined in Control and Raptor KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes. (e) LC3 levels in control and Raptor KO macrophages incubated with vehicle or 100nM Bafilomycin for 2 hours. (f) LC3 intensity (Control: +aa: n = 39; -aa: n = 27; Leu: n = 30; Raptor-KO: +aa: n = 31; -aa: n = 30; Leu: n = 30 cells) and number of puncta (n = 52 cells/group) were analyzed by immunofluorescence microscopy in control and Raptor KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes. (g) Quantification of LC3 intensity by immunofluorescence microscopy in control and ATG5-KO macrophages incubated with regular medium or amino acid-free medium with and without leucine for 30 minutes (Control: +aa: n = 52; -aa: n = 50; Leu: n = 51; ATG5 KO: +aa: n = 36; -aa: n = 35; Leu: n = 47 cells). (h) ATG5-KO macrophages were co-incubated with vehicle or CC ± leucine and percent of caspase 3/7-positive cells were quantified in >103 cells from acquired images (-aa: n = 13; -aa+cc: n = 11; Leu: n = 10; Leu+ cc.: n = 12 image fields). For all graphs, data presented as mean ±SEM. *P < 0.05, one-way ANOVA with Tukey’s test.
