Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Reduced adiposity in Myct1 ecKO mice is independent of angiogenesis, adipogenesis, and systemic metabolic activity. (A) Experimental workflow for monitoring early changes in metabolism of Myct1 ecKO mice. (B) Food intake over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; Welch’s t test, P > 0.05. (C) Total horizontal activity over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (D) RER over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (E) Energy expenditure over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice; mean ± SD; unpaired t test, P > 0.05. (A–E) Results are shown for males at 23°C. Similar results were obtained at 30°C and for females at both temperatures (data not shown). (F) Myct1 ablation does not affect adipose tissue fibrosis. Masson’s trichrome staining of retroperitoneal fat sections of wild-type and Myct1 ecKO mice. Scale bar, 100 μm. (G) Quantification of fibrosis as percentage of area positive for collagen based on Masson’s trichrome stain. n = 6 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (H) Myct1 ablation does not affect the vascular density of WAT. Staining of retroperitoneal sections for Pecam1 (green) and Fabp4 (magenta). Scale bar, 50 µm. (I) Quantification of vascular density as percentage of Pecam1 + adipose tissue area. n = 4 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (J) Quantification of CD45 neg CD31 + cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; Mann–Whitney test, P > 0.05. (K) Experimental workflow for analysis of postnatal retina angiogenesis. (L) Staining of P5 and P7 retina for Pecam1 (black). Scale bar, 1 mm. Magenta circle: outline of the wild-type retina vasculature at the indicated time point; yellow arrow: vascular outgrowth from optic nerve. (M) Quantification of vascular outgrowth for P5 and P7 wild-type and Myct1 ecKO pups. n = 3–6 mice per genotype; mean ± SD; multiple Mann–Whitney tests, P = 0.009 (*) at P5 and P > 0.05 at P7. (N) Quantification of vascular density for P5 and P7 wild-type and Myct1 ecKO pups. n = 3–4 mice per genotype; mean ± SD; multiple Mann–Whitney tests, P > 0.05. (O) Quantification of CD45 + immune cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (P) Quantification of CD45 neg CD31 neg Sca1 + adipocyte progenitor cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (J, O, and P) Data acquired during the same experiments. (Q) Experimental workflow for ex vivo adipogenesis assay. (R) Myct1 ablation does not affect ex vivo adipogenesis. SVF isolated from the inguinal fat pad of wild-type and Myct1 ecKO mice was treated with control or adipogenic cocktail for 4 days. Staining of SVF for perilipin (gray) and DNA (blue). Scale bar, 200 µm. Gonadal SVF provided similar results (data not shown). (S) Quantification of perilipin + area normalized to cell number for SVF. n = 9 mice per condition and genotype; mean ± SD; two-way ANOVA, P > 0.05. (T) Myct1 ablation limits WAT (yellow, adipocytes) expansion independent of angiogenesis (red, vasculature). Icons used in A, K, and T were created with BioRender.com and modified in Affinity. See also .

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Activity Assay, Staining, Flow Cytometry, MANN-WHITNEY, Ex Vivo, Isolation, Control, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Ablation of Myct1 upregulates pathways involved in protein folding, ribonucleoprotein assembly, and mTORC1 signaling. (A) Experimental workflow of the scRNA-seq experiment. CD45 neg CD31 + cells were sorted from mesenteric fat of 12-wk-old wild-type and Myct1 ecKO mice. n = 2 per genotype. (B) UMAP representation of cells retained in the scRNA-seq and colored according to cell type. BECs, blood endothelial cells; LECs, lymphatic endothelial cells; vSMCs, vascular smooth muscle cells. (C) Myct1 is efficiently deleted from ECs in Myct1 ecKO mice. Distribution of Myct1, Pecam1 , and Cdh5 expression (ln[normalized counts +1]) in the BEC cluster in wild-type and Myct1 ecKO cells. (D) GO terms and Hallmark gene sets overrepresented among upregulated (red bars) or downregulated (gray bars) genes in Myct1 ecKO BECs compared with wild-type BECs. (E) Myct1 deficiency in ECs increases protein levels of mTORC1 target Mat2a (methionine adenosyltransferase 2A). Staining of WAT of Myct1 ecKO or wild-type control mice for Mat2a (green) and pan-endothelial marker Erg (magenta). Scale bar, 50 µm. (F) Quantification of data shown in E. n = 8 mice per genotype from three independent experiments. mean ± SD; paired t test, P = 0.0191 (*). Icons used in A were created with BioRender.com and modified in Affinity. See also .

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Expressing, Staining, Control, Marker, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: 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 . Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Knockdown, Cell Culture, Transfection, Control, Staining, Activation Assay, Western Blot, In Vivo, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3, related to Fig. 5 . (A) The MYCT1 protein is highly conserved across vertebrates. An alignment of human MYCT1 protein sequence with those of the indicated species, amino acid conservation is color-coded as indicated in the legend below. Percentages next to species indicate amino acid sequence identity (left) and homology (right) in comparison with human sequence. (B) Short MYCT1 isoform is predominant in ECs. Cells were transduced with Ad- GFP (control) or Ad- MYCT1 (187-aa isoform with C-terminal V5 tag) adenoviruses. Western blot analysis for the indicated proteins. (C) Workflow for mass spectrometry experiments. MYCT1-negative SW480 colon cancer cells were used to exclude nonspecific interactors pulled down by MYCT1 IgG. n = 2 independent experiments. (D) Venn diagram showing how the short list of MYCT1 interactors was selected. (E) Interaction between MYCT1 and IFITM2/3 was analyzed by PLA in ECs. siRNA-mediated knockdown of either protein confirmed specificity of PLA signal. Staining of ECs for PLA dots (gray), VE-cadherin (magenta), and DNA (blue). Scale bar, 50 µm. (F) Quantification of the number of PLA dots per cell in control, MYCT1 KD , and IFITM2/3 KD cells. n = 3 independent experiments; 500–1,500 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons, P = 0.036 (*) for MYCT1 knockdown effect. (G) Positive control (VE-cadherin::β-catenin) and negative control (MYCT1 antibody alone) for PLA signal in human brain sections. PLA dots (gray) and staining of ECs for VE-cadherin or Pecam1 (magenta) and DNA (blue). Arrowhead, colocalization of PLA dots with vascular marker. Scale bar, 20 µm. Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Sequencing, Comparison, Transduction, Control, Western Blot, Mass Spectrometry, Knockdown, Staining, Positive Control, Negative Control, Marker

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3. (A) Endogenous MYCT1 is located at cell–cell junctions (arrow) and in puncta (arrowhead). Staining of human primary ECs for MYCT1 (black), VE-cadherin (magenta), and DNA (blue). Scale bar, 10 µm. (B) MYCT1 is a membrane protein. Western blot analysis of various EC fractions for MYCT1, GAPDH, PECAM1, H3K27ac, and vimentin proteins. Cy, cytoplasm; Mb, membrane; Nu, nucleus; Ck, cytoskeleton. (C) MYCT1 is glycosylated. Western blot analysis of MYCT1 protein electrophoretic mobility in control and PNGase-F–treated lysates. (D) Schematic model of MYCT1 structure and domains with phosphorylation sites, identified by mass spectrometry. MYCT1 phosphorylation sites are highly conserved as indicated by the color scale. Asterisks indicate sites also described at https://www.phosphosite.org/ . (E) Top five proteins interacting with MYCT1 as identified by mass spectrometry, among which IFITM2 and IFITM3. ECs were transduced with recombinant adenoviruses to transiently overexpress MYCT1 or GFP, as a control. Cell lysates were collected 48 h after transduction, immunoprecipitated using MYCT1 antibody or a control IgG, and analyzed by mass spectrometry. Proteins interacting with both endogenous and overexpressed MYCT1 were selected and ranked by normalized spectral abundance factor (NSAF) from two independent mass spectrometry (MS) experiments are shown (31 proteins); the top five proteins are highlighted in magenta. (F) GO terms of the cellular component and biological process overrepresented in the MYCT1 interactome. Fisher’s exact test with adjustment for false discovery rate (FDR). (G) Validation of IFITM2/3 and MYCT1 interaction by co-IP. EC lysates from confluent ECs were immunoprecipitated (IP) with MYCT1 or control IgG and blotted for IFITM2/3. H, IgG heavy chain; L, IgG light chain. (H) IFITM2/3 are constitutively expressed in ECs in vitro and in vivo . Staining of human primary ECs (upper panels), human brain and WAT sections (lower panels) for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm (brain) and 50 µm (adipose tissue). (I) MYCT1 and IFITM2/3 interact in brain ECs. Proximity ligation assay (PLA) in human brain sections. Detection of PLA dots (gray) in ECs and staining for VE-cadherin (magenta) and DNA (blue). Arrowheads, colocalization of MYCT1::IFITM2/3 PLA dots and VE-cadherin staining. Scale bar, 20 µm. See also . Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Staining, Membrane, Western Blot, Control, Phospho-proteomics, Mass Spectrometry, Transduction, Recombinant, Immunoprecipitation, Biomarker Discovery, Co-Immunoprecipitation Assay, In Vitro, In Vivo, Proximity Ligation Assay

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Endothelial-specific activation of mTORC1 signaling recapitulates adipose tissue phenotype of Myct1 ecKO mice. (A) Tsc1 ecKO mouse model. See Materials and methods for details. (B) Endothelial Tsc1 ablation activates sustained mTORC1 signaling in vivo . En face staining of aorta for p-S6 (gray) and VE-cadherin (magenta). Scale bar, 50 μm. (C) Quantification of mTORC1 activation in the aortic endothelium of wild-type and Tsc1 ecKO mice. The percentage of p-S6 + cells was quantified in n = 5 mice per genotype and conditions; 300–500 cells were analyzed per aorta; mean ± SD; Welch’s t test, P = 0.0121 (*). (D) Workflow of Tsc1 ecKO mouse analysis. (E) Wild-type and Tsc1 ecKO mice were analyzed before significant difference in body weight. Quantification of body weight at the start and end of experiment. n = 10–12 mice per genotype; mean ± SD; multiple unpaired t tests, P > 0.05 at start, P = 0.116 at end. (F) Quantification of fat pad weight to body weight ratio relative to wild-type mice. n = 9 mice per genotype; mean ± SD; multiple unpaired t tests, P = 0.001 (*) for interscapular WAT (IsWAT), P = 0.027 for interscapular BAT (IsBAT), P < 0.001 (*) for inguinal (Ing), P < 0.001 (*) for retroperitoneal (RP), P < 0.001 (*) for gonadal (Gon), and P < 0.001 (*) for mesenteric (Mes). (G) Tsc1 ablation reduces size of adipocytes. Retroperitoneal thick sections stained for Laminin α4 (gray) and Pecam1 (magenta). Scale bar, 50 μm. (H) Quantification of adipocyte size in wild-type and Tsc1 ecKO fat. n = 5 mice per genotype; mean ± SD; unpaired t test, P = 0.0056 (*). (I) Schematic view of the role of endothelial MYCT1–IFITM2/3 complexes in WAT homeostasis. MYCT1 interacts with IFITM2/3, limiting their function and allowing nutrient transport for energy storage. In the absence of MYCT1, IFITM2/3 accumulate in early endosomes and trigger continuous endolysosomal cargo degradation and hyperactivation of mTORC1 signaling in ECs, restricting energy storage in WAT. Icons used in A, D, and I were created with BioRender.com and modified in Affinity.

Article Snippet: Pecam1 (mouse) forward , Eurofins , 5′-AAC AGA AAC CCG TGG AGA TG-3′.

Techniques: Activation Assay, In Vivo, Staining, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Reduced adiposity in Myct1 ecKO mice is independent of angiogenesis, adipogenesis, and systemic metabolic activity. (A) Experimental workflow for monitoring early changes in metabolism of Myct1 ecKO mice. (B) Food intake over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; Welch’s t test, P > 0.05. (C) Total horizontal activity over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (D) RER over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (E) Energy expenditure over 24 h (23°C) is similar between wild-type and Myct1 ecKO mice. n = 10 mice; mean ± SD; unpaired t test, P > 0.05. (A–E) Results are shown for males at 23°C. Similar results were obtained at 30°C and for females at both temperatures (data not shown). (F) Myct1 ablation does not affect adipose tissue fibrosis. Masson’s trichrome staining of retroperitoneal fat sections of wild-type and Myct1 ecKO mice. Scale bar, 100 μm. (G) Quantification of fibrosis as percentage of area positive for collagen based on Masson’s trichrome stain. n = 6 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (H) Myct1 ablation does not affect the vascular density of WAT. Staining of retroperitoneal sections for Pecam1 (green) and Fabp4 (magenta). Scale bar, 50 µm. (I) Quantification of vascular density as percentage of Pecam1 + adipose tissue area. n = 4 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (J) Quantification of CD45 neg CD31 + cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; Mann–Whitney test, P > 0.05. (K) Experimental workflow for analysis of postnatal retina angiogenesis. (L) Staining of P5 and P7 retina for Pecam1 (black). Scale bar, 1 mm. Magenta circle: outline of the wild-type retina vasculature at the indicated time point; yellow arrow: vascular outgrowth from optic nerve. (M) Quantification of vascular outgrowth for P5 and P7 wild-type and Myct1 ecKO pups. n = 3–6 mice per genotype; mean ± SD; multiple Mann–Whitney tests, P = 0.009 (*) at P5 and P > 0.05 at P7. (N) Quantification of vascular density for P5 and P7 wild-type and Myct1 ecKO pups. n = 3–4 mice per genotype; mean ± SD; multiple Mann–Whitney tests, P > 0.05. (O) Quantification of CD45 + immune cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (P) Quantification of CD45 neg CD31 neg Sca1 + adipocyte progenitor cells as percentage of total cells in fat pad as measured by flow cytometry. n = 7–8 mice per genotype; mean ± SD; unpaired t test, P > 0.05. (J, O, and P) Data acquired during the same experiments. (Q) Experimental workflow for ex vivo adipogenesis assay. (R) Myct1 ablation does not affect ex vivo adipogenesis. SVF isolated from the inguinal fat pad of wild-type and Myct1 ecKO mice was treated with control or adipogenic cocktail for 4 days. Staining of SVF for perilipin (gray) and DNA (blue). Scale bar, 200 µm. Gonadal SVF provided similar results (data not shown). (S) Quantification of perilipin + area normalized to cell number for SVF. n = 9 mice per condition and genotype; mean ± SD; two-way ANOVA, P > 0.05. (T) Myct1 ablation limits WAT (yellow, adipocytes) expansion independent of angiogenesis (red, vasculature). Icons used in A, K, and T were created with BioRender.com and modified in Affinity. See also .

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Activity Assay, Staining, Flow Cytometry, MANN-WHITNEY, Ex Vivo, Isolation, Control, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Ablation of Myct1 upregulates pathways involved in protein folding, ribonucleoprotein assembly, and mTORC1 signaling. (A) Experimental workflow of the scRNA-seq experiment. CD45 neg CD31 + cells were sorted from mesenteric fat of 12-wk-old wild-type and Myct1 ecKO mice. n = 2 per genotype. (B) UMAP representation of cells retained in the scRNA-seq and colored according to cell type. BECs, blood endothelial cells; LECs, lymphatic endothelial cells; vSMCs, vascular smooth muscle cells. (C) Myct1 is efficiently deleted from ECs in Myct1 ecKO mice. Distribution of Myct1, Pecam1 , and Cdh5 expression (ln[normalized counts +1]) in the BEC cluster in wild-type and Myct1 ecKO cells. (D) GO terms and Hallmark gene sets overrepresented among upregulated (red bars) or downregulated (gray bars) genes in Myct1 ecKO BECs compared with wild-type BECs. (E) Myct1 deficiency in ECs increases protein levels of mTORC1 target Mat2a (methionine adenosyltransferase 2A). Staining of WAT of Myct1 ecKO or wild-type control mice for Mat2a (green) and pan-endothelial marker Erg (magenta). Scale bar, 50 µm. (F) Quantification of data shown in E. n = 8 mice per genotype from three independent experiments. mean ± SD; paired t test, P = 0.0191 (*). Icons used in A were created with BioRender.com and modified in Affinity. See also .

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Expressing, Staining, Control, Marker, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: 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 . Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Knockdown, Cell Culture, Transfection, Control, Staining, Activation Assay, Western Blot, In Vivo, Modification

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3, related to Fig. 5 . (A) The MYCT1 protein is highly conserved across vertebrates. An alignment of human MYCT1 protein sequence with those of the indicated species, amino acid conservation is color-coded as indicated in the legend below. Percentages next to species indicate amino acid sequence identity (left) and homology (right) in comparison with human sequence. (B) Short MYCT1 isoform is predominant in ECs. Cells were transduced with Ad- GFP (control) or Ad- MYCT1 (187-aa isoform with C-terminal V5 tag) adenoviruses. Western blot analysis for the indicated proteins. (C) Workflow for mass spectrometry experiments. MYCT1-negative SW480 colon cancer cells were used to exclude nonspecific interactors pulled down by MYCT1 IgG. n = 2 independent experiments. (D) Venn diagram showing how the short list of MYCT1 interactors was selected. (E) Interaction between MYCT1 and IFITM2/3 was analyzed by PLA in ECs. siRNA-mediated knockdown of either protein confirmed specificity of PLA signal. Staining of ECs for PLA dots (gray), VE-cadherin (magenta), and DNA (blue). Scale bar, 50 µm. (F) Quantification of the number of PLA dots per cell in control, MYCT1 KD , and IFITM2/3 KD cells. n = 3 independent experiments; 500–1,500 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons, P = 0.036 (*) for MYCT1 knockdown effect. (G) Positive control (VE-cadherin::β-catenin) and negative control (MYCT1 antibody alone) for PLA signal in human brain sections. PLA dots (gray) and staining of ECs for VE-cadherin or Pecam1 (magenta) and DNA (blue). Arrowhead, colocalization of PLA dots with vascular marker. Scale bar, 20 µm. Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Sequencing, Comparison, Transduction, Control, Western Blot, Mass Spectrometry, Knockdown, Staining, Positive Control, Negative Control, Marker

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3. (A) Endogenous MYCT1 is located at cell–cell junctions (arrow) and in puncta (arrowhead). Staining of human primary ECs for MYCT1 (black), VE-cadherin (magenta), and DNA (blue). Scale bar, 10 µm. (B) MYCT1 is a membrane protein. Western blot analysis of various EC fractions for MYCT1, GAPDH, PECAM1, H3K27ac, and vimentin proteins. Cy, cytoplasm; Mb, membrane; Nu, nucleus; Ck, cytoskeleton. (C) MYCT1 is glycosylated. Western blot analysis of MYCT1 protein electrophoretic mobility in control and PNGase-F–treated lysates. (D) Schematic model of MYCT1 structure and domains with phosphorylation sites, identified by mass spectrometry. MYCT1 phosphorylation sites are highly conserved as indicated by the color scale. Asterisks indicate sites also described at https://www.phosphosite.org/ . (E) Top five proteins interacting with MYCT1 as identified by mass spectrometry, among which IFITM2 and IFITM3. ECs were transduced with recombinant adenoviruses to transiently overexpress MYCT1 or GFP, as a control. Cell lysates were collected 48 h after transduction, immunoprecipitated using MYCT1 antibody or a control IgG, and analyzed by mass spectrometry. Proteins interacting with both endogenous and overexpressed MYCT1 were selected and ranked by normalized spectral abundance factor (NSAF) from two independent mass spectrometry (MS) experiments are shown (31 proteins); the top five proteins are highlighted in magenta. (F) GO terms of the cellular component and biological process overrepresented in the MYCT1 interactome. Fisher’s exact test with adjustment for false discovery rate (FDR). (G) Validation of IFITM2/3 and MYCT1 interaction by co-IP. EC lysates from confluent ECs were immunoprecipitated (IP) with MYCT1 or control IgG and blotted for IFITM2/3. H, IgG heavy chain; L, IgG light chain. (H) IFITM2/3 are constitutively expressed in ECs in vitro and in vivo . Staining of human primary ECs (upper panels), human brain and WAT sections (lower panels) for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm (brain) and 50 µm (adipose tissue). (I) MYCT1 and IFITM2/3 interact in brain ECs. Proximity ligation assay (PLA) in human brain sections. Detection of PLA dots (gray) in ECs and staining for VE-cadherin (magenta) and DNA (blue). Arrowheads, colocalization of MYCT1::IFITM2/3 PLA dots and VE-cadherin staining. Scale bar, 20 µm. See also . Source data are available for this figure: .

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Staining, Membrane, Western Blot, Control, Phospho-proteomics, Mass Spectrometry, Transduction, Recombinant, Immunoprecipitation, Biomarker Discovery, Co-Immunoprecipitation Assay, In Vitro, In Vivo, Proximity Ligation Assay

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: Endothelial-specific activation of mTORC1 signaling recapitulates adipose tissue phenotype of Myct1 ecKO mice. (A) Tsc1 ecKO mouse model. See Materials and methods for details. (B) Endothelial Tsc1 ablation activates sustained mTORC1 signaling in vivo . En face staining of aorta for p-S6 (gray) and VE-cadherin (magenta). Scale bar, 50 μm. (C) Quantification of mTORC1 activation in the aortic endothelium of wild-type and Tsc1 ecKO mice. The percentage of p-S6 + cells was quantified in n = 5 mice per genotype and conditions; 300–500 cells were analyzed per aorta; mean ± SD; Welch’s t test, P = 0.0121 (*). (D) Workflow of Tsc1 ecKO mouse analysis. (E) Wild-type and Tsc1 ecKO mice were analyzed before significant difference in body weight. Quantification of body weight at the start and end of experiment. n = 10–12 mice per genotype; mean ± SD; multiple unpaired t tests, P > 0.05 at start, P = 0.116 at end. (F) Quantification of fat pad weight to body weight ratio relative to wild-type mice. n = 9 mice per genotype; mean ± SD; multiple unpaired t tests, P = 0.001 (*) for interscapular WAT (IsWAT), P = 0.027 for interscapular BAT (IsBAT), P < 0.001 (*) for inguinal (Ing), P < 0.001 (*) for retroperitoneal (RP), P < 0.001 (*) for gonadal (Gon), and P < 0.001 (*) for mesenteric (Mes). (G) Tsc1 ablation reduces size of adipocytes. Retroperitoneal thick sections stained for Laminin α4 (gray) and Pecam1 (magenta). Scale bar, 50 μm. (H) Quantification of adipocyte size in wild-type and Tsc1 ecKO fat. n = 5 mice per genotype; mean ± SD; unpaired t test, P = 0.0056 (*). (I) Schematic view of the role of endothelial MYCT1–IFITM2/3 complexes in WAT homeostasis. MYCT1 interacts with IFITM2/3, limiting their function and allowing nutrient transport for energy storage. In the absence of MYCT1, IFITM2/3 accumulate in early endosomes and trigger continuous endolysosomal cargo degradation and hyperactivation of mTORC1 signaling in ECs, restricting energy storage in WAT. Icons used in A, D, and I were created with BioRender.com and modified in Affinity.

Article Snippet: Pecam1 (mouse) reverse , Eurofins , 5′-GTC TCT GTG GCT CTC GTT CC-3′.

Techniques: Activation Assay, In Vivo, Staining, Modification