Figure S3.
MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45negCD31+) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H)MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKTSer473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKTThr308 levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M)n = 5 independent experiments; paired t tests. (N)MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P)MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: SourceData FS3. Refer to the image caption for details. The image contains multiple panels illustrating the impact of endothelial M Y C T 1 ablation on adiposity. Panel A shows a flow cytometry plot with C D 45-P E and C D 31-B V 421 axes, indicating the gating strategy for sorting endothelial cells from mesenteric fat. Panel B presents a dot plot of markers for various cell clusters, with dot color representing scaled average expression and dot sizes denoting the percent of cells expressing each gene. Panel C is a table listing the number of differentially expressed genes between M y c t 1 e c K O and wildtype cell clusters. Panel D features a volcano plot of D E G s between wildtype and M y c t 1 e c K O mice in the B E C cluster, highlighting genes more expressed in each group. Panel E displays western blot results for M Y C T 1 protein levels in different human primary endothelial cells. Panel F shows M Y C T 1 and D N A staining in endothelial cells, with arrows indicating M Y C T 1-positive cells. Panel G presents western blot analysis of M Y C T 1 expression after M Y C T 1 s i R N A knockdown and control treatment. Panel H includes western blot results for proteins involved in m T O R C 1 signaling, showing increased phosphorylation of S 6 upon M Y C T 1 knockdown. Panels I to L quantify the levels of phosphorylated proteins normalized to their total forms. Panel M quantifies M Y C T 1 levels normalized to vinculin. Panel N shows western blot results for p 70 slash S 6 K phosphorylation in response to amino acids. Panel O quantifies the data from Panel N. Panel P displays cell staining for p-S 6, VE-cadherin, and D N A under different stimulation conditions.

MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45negCD31+) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H)MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKTSer473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKTThr308 levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M)n = 5 independent experiments; paired t tests. (N)MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P)MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: SourceData FS3.

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