Fig. 3

AIMTOR responses to specific mTOR signaling cues. a, b AIMTOR BRET intensity increases in response to amino acid stimulation. a AIMTOR BRET intensities in C2C12 cells fasted 3 h in medium lacking four amino acids (leucine, methionine, lysine, and glutamine) then incubated 1 h with the same medium, or medium supplemented with leucine, rapamycin, or with leucine, methionine, lysine, and glutamine (“- 4 AAs + Rich”). Histogram: mean ± SEM of BRET intensity, with BRET intensity from “- 4AAs” condition subtracted from the indicated condition. n = 3–6 independent experiments. One-way ANOVA repeated measures, post hoc analysis with Tukey’s multiple comparison test: ***p < 0.001 compared to “- 4 AAs” condition, ^###p < 0.001 compared to “- 4 AAs + Leucine” condition. b AIMTOR BRET intensities in human primary muscle cells incubated in “rich” medium (white circles) or fasted 3 h in medium lacking four amino acids and then treated for 1 h in the same medium (gray circles) or in rich medium (black circles). Dots represent single experiments; horizontal bar shows the mean of 3 independent experiments. One-way ANOVA repeated measures, post hoc analysis with Tukey’s multiple comparison test: ***p < 0.001 compared to “rich “condition and ^###p < 0.001 compared to “- 4 AAs” condition. c Insulin increases AIMTOR BRET intensity: mean ± SEM of BRET intensity of AIMTOR-transfected C2C12 cell serum fasted overnight and stimulated or not with insulin for 1 h (n = 6), with BRET intensity from the “O/N serum fasted” condition subtracted from each condition. Mann-Whitney t test, ***p < 0.001. d Silencing of REDD1, a negative regulator of mTORC1, increases AIMTOR BRET intensity. Dots represent single experiments of H1299 cells expressing AIMTOR previously transfected with a siRNA targeting REDD1 (black colored circles) or a non-targeting control siRNA (white colored circles). Horizontal bar: mean BRET intensity of 3 independent transfections. Wilcoxon two-tailed matched paired sign rank test, **p < 0.01