Fig. 2

AIMTOR responses to phosphatase and mTOR inhibitors. a–c AIMTOR reports phosphorylation-dependent events, triggered by mTOR. Mean BRET intensities recorded in H1299 cells expressing AIMTOR. Cells were incubated for 90 min or overnight (O/N) either with a mix of serine/threonine phosphatase inhibitors (a), the mTOR inhibitor rapamycin (b), and Torin (c) or with the DMSO vehicle as control. Each dot on the graphs represents the BRET intensity of one experiment and the horizontal bar is the mean of BRET intensities recorded in 3 independent experiments. Grouped analysis, 2-way ANOVA (“time” and “treatment”) with post hoc using the Bonferroni multiple comparison test. **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the respective DMSO (control) condition. ^##p < 0.01 and ^####p < 0.0001 compared to the 25 nM condition. ^$$p < 0.01 compared to the 100-nM condition. The interaction between “time” and “treatment” is significant in a (p = 0.0006) and non-significant in b (p = 0.0959) and significant in c (p < 0.0001). d, e BRET imaging of mTORC1 activities detected with AIMTOR. Biosensor-transfected C2C12 cells were treated overnight with rapamycin (250 nM) or DMSO (control). Reporter expression is reported by YPET expression (GFP) and NLuc emission (Em450), whereas the excitation of YPET by the non-radiative transfer of energy from NLuc is recorded in the Em535 image. Pixel-by-pixel division of the Em 535 and Em 450 images gives rise to the pseudo-colored BRET image (Em 535/Em 450). Representative images (d) and quantification of the average 535nm/450nm ratio intensities (e) of AIMTOR and mutant AIMTOR-T757A signals in transfected proliferating C2C12 myoblasts. Each dot in the graph represents the BRET intensity recording of one cell, and the horizontal bar shows the mean obtained from 20 to 25 cells per condition. Mann-Whitney statistical analysis with ****p < 0.0001 compared to the AIMTOR control condition