Fig. 5

AIMTOR reports mTORC1 activity in differentiating muscle cells. a mTORC1 activity in C2C12 differentiating cells (triplicates wells for each stage). Bands show phosphorylated S6K1 (above), Myogenin, a differentiation marker (middle) and total protein load revealed (below). Right: quantification of phosphorylated S6K1 bands normalized by total protein load. One-way ANOVA, multiple comparison Tukey’s post hoc test. **p < 0.01 compared to D0 and ^###p < 0.001 compared to D1. b BRET imaging of 5-day differentiated C2C12 expressing cytosolic AIMTOR treated overnight with rapamycin or DMSO (control). Right: BRET intensity of individual cells treated with DMSO or rapamycin; horizontal bars show mean BRET intensity calculated from 20 to 25 cells per condition. Mann-Whitney statistical analysis with ****p < 0.0001 compared to control condition. c, d Mean BRET intensity of differentiating C2C12 cells expressing AIMTOR localized to various subcellular compartments: each dot represents the mean BRET intensity of one experiment. In c, BRET intensity recorded in cells in their proliferative phase (D0), at day 1 (D1), day 3 (D3), and day 5 (D5) of differentiation and expressing, cytosolic (NES), lysosomal, nuclear, mitochondrial AIMTOR or the T757A non-responding mutant. One-way ANOVA non-parametric Friedman test with repeated measure and multiple comparison Dunn’s post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001 and compared to D0. ^#p < 0.05 compared to D3. In d, BRET intensity in 5-day differentiated C2C12, fasted with medium lacking four amino acids containing 2% or 10% of dialyzed serum, and stimulated (gray circles) or not (white circles) with leucine during 1 h. Grouped analysis, two-way ANOVA (“leucine treatment” and “serum concentration”) with post hoc using the Bonferroni multiple comparison test. *p < 0.05 and ***p < 0.001 compared to the fasting condition. Grouped analysis also performed for cytosolic and lysosomal AIMTOR, two-way ANOVA (“Biosensor Localization” and “serum concentration”). The interaction is significant (p = 0.0269). ^##p < 0.01 compared to the 2% serum condition