MYCT1 limits endothelial mTORC1 signaling. (A) MYCT1 knockdown in quiescent ECs hyperactivates mTORC1 signaling. Confluent ECs were cultured in complete medium for 1.5 days after siRNA transfection and treated overnight with 10 nM mTORC1 inhibitor rapamycin or DMSO as a control. Staining for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. (B) Quantification of mTORC1 activation in control and MYCT1^KD cells in the presence or absence of rapamycin. The percentage of p-S6⁺ cells was quantified in the indicated conditions. n = 6 independent experiments (3 in the presence of rapamycin); 500–5,000 cells were analyzed per condition for each experiment; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.011 (*) for MYCT1 knockdown effect in control conditions and P < 0.0001 (*) for rapamycin effect on MYCT1^KD cells. (C) MYCT1 limits protein synthesis. Confluent control and MYCT1^KD ECs were cultured in complete medium for 2 days after siRNA transfection and treated for 50 min with 10 µM OPP. Staining for OPP (gray), VE-cadherin (magenta), and DNA (blue). Arrowhead, OPP puncta. Scale bar, 20 µm. (D) Quantification of OPP puncta per cell in control and MYCT1^KD cells. n = 3 independent experiments; 20–40 cells analyzed per condition for each experiment; mean ± SD; Welch’s t test, P = 0.042 (*). (E)MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. Confluent ECs were starved for 1 h and restimulated with amino acids for 30 min. Western blot analysis for the indicated proteins. (F)MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. Staining for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. (G) Quantification of mTORC1 activation in response to amino acid supplementation. The percentage of p-S6⁺ cells was quantified in the indicated conditions. n = 4 independent experiments; 150–500 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.002 (*) for MYCT1 knockdown effect under amino acid supplementation. (H)Myct1 ablation hyperactivates mTORC1 signaling in vivo in a feeding status–dependent manner. Wild-type and Myct1^ecKO mice were starved overnight or fed ad libitum. En face staining of aorta for p-S6 (gray) and VE-cadherin (magenta). Scale bar, 50 μm. (I) Quantification of mTORC1 activation in the aortic endothelium of wild-type and Myct1^ecKO mice, starved or fed ad libitum. The percentage of p-S6⁺ cells was quantified in n = 4–6 mice per genotype and conditions; 300–500 cells were analyzed per aorta; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.026 (*) for Myct1 ablation effect in fed mice. (J)Myct1 ablation hyperactivates mTORC1 signaling in ECs of the retroperitoneal fat pad. Mice fed ad libitum. Staining for p-S6 (gray/black), endomucin (magenta), and DNA (blue). Arrowheads, high endothelial p-S6⁺ signal. Scale bar, 20 μm. (K) Quantification of p-S6 intensity in ECs of the retroperitoneal fat pad. p-S6 intensity was quantified in endomucin⁺ (EMCN⁺) area and normalized to wild type. n = 6 mice per condition; mean ± SD; paired t test, P = 0.021 (*). (L)Myct1 ablation hyperactivates mTORC1 signaling in capillary ECs of the mesenteric fat pad. Mice fed ad libitum. Whole-mount staining for p-S6 (gray/black) and Pecam1 (magenta). Scale bar, 20 μm. (M) Schematic view of MYCT1 limitation of mTORC1 signaling in ECs. MYCT1 ablation hyperactivates mTORC1 signaling in response to amino acids and nutrients. Icons used in M were created with BioRender.com and modified in Affinity. See also Fig. S3, E–P. Source data are available for this figure: SourceData F4.