mTORC1 signaling is activated in the vicinity of FAs. (A) Fluorescence intensity line profile plots corresponding to lines exemplified by an arrow in Fig. 3 A. (B) U2OS, HEK293, and Cos7 cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) and immunostained for p-S6 and the FA protein paxillin. Colocalization (Manders coefficient) between p-S6 and paxillin was analyzed. (C) Representative confocal image of GFP-paxillin intensity-scaled ratio image of TORCAR FRET-based biosensor in serum-starved HeLa cells before and after stimulation with 25 mM membrane-permeable leucine methylester. Note that, to visualize the differences, the images were gamma adjusted, and black borders in zoom insets highlight the ROIs corresponding to FAs. (D) Quantification of ratio change (percentage) of TORCAR biosensor signal before stimulation (−FCS), after stimulation with FCS (+FCS), in the presence of rapamycin (+FCS +Rapamycin), and in response to leucine methylester (+Leucine). Each data point represents a coverslip including several cells; box-and-whisker plot whiskers represent minimum and maximum values. Error bars represent SEM; n = 3 independent experiments. ****, P < 0.0001; two-way ANOVA with Sidak correction. For B, **, P < 0.01, ***, P < 0.001 two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.