Extended Data Fig. 4: -for Main Fig. 4.
Extended Data Fig. 4 (A) Representative images of cultured human monocytes-derived macrophages (HMDMs) from isolated human circulating monocytes before and after 12 days of differentiation. The picture represents for three independent experiments. (B-C) FACS (B) and immunofluorescence (C) analysis of differentiated HMDMs by macrophage markers CD11b, CD18 and CD68. The picture represents for three independent experiments. (D) Western blot analysis of mTOR activation in HMDM (using phospho-S6 and –S6K) after treatment with leucine (2 mM) or full amino acids. The picture represents for three independent experiments. (E-F) Immunofluorescence microscopy analysis and quantification of mTORC1 activation (using phospho-S6) (E) and autophagy inhibition (using LC3 puncta formation) (F) in HMDMs treated with 2 mM leucine. The IF data (E and F) were obtained from three independent experiments with analysis of n ≥ 15 cells per experiment (n = 45 in Group –aa and n = 40 in Group Leu for E; n = 58 in Group –aa and n = 58 in Group Leu for F). P < 10e-8 for E and F. (G) Representative images of LC3 in HMDMs for Fig. 5d. (H) Immunoblot analysis of the phosphorylation of AMPK and ULK1 in HMDMs by the selected amino acids (applied at 2 mM). n = 4 independent experiments per group. P = 0.00132 and determined by One-way ANOVA with Dunnett’s multiple comparisons. (I) Representative images of LC3 in HMDMs for Fig. 5g. For all graphs, data are presented as mean ± SEM, **P < 0.01 and ***P < 0.001.
