Figure 6.
MYCT1 limits enlargement of IFITM2/3+ endosomes. (A) MYCT1 and IFITM2/3 display inverse regulatory dynamics. MYCT1 knockdown increases total IFITM2/3 protein levels, whereas IFITM2/3 knockdown reduces total MYCT1 protein levels. Western blot analysis of confluent ECs 48 h after siRNA transfection for the indicated proteins. Quantification of total MYCT1 and IFITM2/3 protein levels relative to the control is indicated as the average of all experiments under the respective blots. n = 4 independent experiments; one-way ANOVA with Dunnett’s multiple comparisons test; P < 0.0001 (*) for MYCT1 knockdown effect on MYCT1, P = 0.004 (*) for IFITM2/3 knockdown effect on MYCT1, P = 0.015 (*) for MYCT1 knockdown effect on IFITM2/3, and P = 0.0016 (*) for IFITM2/3 knockdown effect on IFITM2/3. (B)MYCT1 knockdown increases IFITM2/3+ vesicle volume. Staining of ECs for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Yellow box: magnification of IFITM2/3 from highlighted areas, shown below. Scale bar, 20 µm. (C) Quantification of the IFITM2/3+ vesicle volume in control and MYCT1KD cells. n = 4 independent experiments; 500–1,500 vesicles from 15 to 20 cells were analyzed per condition for each experiment; mean ± SD; unpaired t test; P = 0.039 (*). (D) IFITM2/3 localize mainly to the RAB5+ early endosome in ECs, rather than to RAB7+ late endosome and LAMP1+ lysosome. Staining for IFITM2/3 (gray), RAB5 or RAB7 or LAMP1 (green), and DNA (blue). Yellow box: magnification of IFITM2/3 and endolysosomal markers from highlighted areas, shown below. Yellow arrows, IFITM2/3+RAB5+ vesicles; magenta arrows, IFITM2/3+RAB7neg or IFITM2/3+LAMP1neg vesicles. Scale bar, 20 µm. (E) Quantification of colocalization of IFITM2/3 with RAB5, RAB7, and LAMP1 by Manders’ overlap coefficients. n = 2–4 independent experiments; 15–25 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P < 0.001 (*) for both RAB5 versus RAB7 and RAB5 versus LAMP1. (F)MYCT1 knockdown causes enlargement of RAB5+ early endosomes. Staining of ECs for RAB5 (gray/black), VE-cadherin (magenta), and DAPI (blue). Cyan box: magnification of RAB5 from highlighted areas, shown below. Scale bar, 10 μm. (G) Quantification of RAB5+ vesicle volume. n = 4 independent experiments; 400–800 vesicles were analyzed per condition for each experiment; mean ± SD; unpaired t test, P = 0.0133 (*). (H)Myct1 ablation enlarges Rab5+ early endosomes in aortic ECs. En face staining of aorta from wild-type or Myct1ecKO mice for Rab5 (gray/black) and VE-cadherin (magenta). Cyan box: magnification of RAB5 from highlighted areas, shown below. Scale bar, 5 μm. (I) Quantification of Rab5+ vesicle volume in aortic ECs. n = 5 mice per genotype; 1,000–4,000 vesicles were analyzed per mouse; mean ± SD; unpaired t test, P = 0.0128 (*). (J)MYCT1 depletion leads to IFITM2/3+ early endosome enlargement. Icons used in J were created with BioRender.com and modified in Affinity. See also Fig. S5, A–G. Source data are available for this figure: SourceData F6. Refer to the image caption for details. The image contains multiple panels showing various experimental results related to the interaction between M Y C T 1 and I F I T M 2 slash 3 proteins. Panel A shows a Western blot analysis with corresponding quantification graphs, indicating the inverse regulatory dynamics between M Y C T 1 and I F I T M 2 slash 3. The graphs display protein levels relative to control, with significant changes noted in M Y C T 1 and I F I T M 2 slash 3 levels upon knockdown of the respective proteins. Panel B presents immunofluorescence images of endothelial cells stained for M Y C T 1, I F I T M 2 slash 3, V E-cadherin, and D N A, with a magnification of I F I T M 2 slash 3 vesicles. Panel C quantifies the I F I T M 2 slash 3 vesicle volume, showing a significant increase in M Y C T 1 knockdown cells. Panel D shows immunofluorescence images of I F I T M 2 slash 3 localization with early endosome marker R A B 5, late endosome marker R A B 7, and lysosome marker LAMP 1, with magnifications highlighting co-localization. Panel E quantifies the co-localization of I F I T M 2 slash 3 with these markers, indicating a primary localization to R A B 5-positive early endosomes. Panel F presents immunofluorescence images of R A B 5 and V E-cadherin in control and M Y C T 1 knockdown cells, with a magnification of R A B 5 vesicles. Panel G quantifies the R A B 5 vesicle volume, showing a significant increase in M Y C T 1 knockdown cells. Panel H shows immunofluorescence images of R A B 5 and V E-cadherin in aortic endothelial cells from wildtype and M y c t 1 e c K O mice, with a magnification of R A B 5 vesicles. Panel I quantifies the R A B 5 vesicle volume in these cells, showing a significant increase in M y c t 1 e c K O mice. Panel J provides a schematic diagram illustrating the effect of M Y C T 1 depletion on I F I T M 2 slash 3-positive early endosome enlargement.

MYCT1 limits enlargement of IFITM2/3+ endosomes. (A) MYCT1 and IFITM2/3 display inverse regulatory dynamics. MYCT1 knockdown increases total IFITM2/3 protein levels, whereas IFITM2/3 knockdown reduces total MYCT1 protein levels. Western blot analysis of confluent ECs 48 h after siRNA transfection for the indicated proteins. Quantification of total MYCT1 and IFITM2/3 protein levels relative to the control is indicated as the average of all experiments under the respective blots. n = 4 independent experiments; one-way ANOVA with Dunnett’s multiple comparisons test; P < 0.0001 (*) for MYCT1 knockdown effect on MYCT1, P = 0.004 (*) for IFITM2/3 knockdown effect on MYCT1, P = 0.015 (*) for MYCT1 knockdown effect on IFITM2/3, and P = 0.0016 (*) for IFITM2/3 knockdown effect on IFITM2/3. (B)MYCT1 knockdown increases IFITM2/3+ vesicle volume. Staining of ECs for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Yellow box: magnification of IFITM2/3 from highlighted areas, shown below. Scale bar, 20 µm. (C) Quantification of the IFITM2/3+ vesicle volume in control and MYCT1KD cells. n = 4 independent experiments; 500–1,500 vesicles from 15 to 20 cells were analyzed per condition for each experiment; mean ± SD; unpaired t test; P = 0.039 (*). (D) IFITM2/3 localize mainly to the RAB5+ early endosome in ECs, rather than to RAB7+ late endosome and LAMP1+ lysosome. Staining for IFITM2/3 (gray), RAB5 or RAB7 or LAMP1 (green), and DNA (blue). Yellow box: magnification of IFITM2/3 and endolysosomal markers from highlighted areas, shown below. Yellow arrows, IFITM2/3+RAB5+ vesicles; magenta arrows, IFITM2/3+RAB7neg or IFITM2/3+LAMP1neg vesicles. Scale bar, 20 µm. (E) Quantification of colocalization of IFITM2/3 with RAB5, RAB7, and LAMP1 by Manders’ overlap coefficients. n = 2–4 independent experiments; 15–25 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P < 0.001 (*) for both RAB5 versus RAB7 and RAB5 versus LAMP1. (F)MYCT1 knockdown causes enlargement of RAB5+ early endosomes. Staining of ECs for RAB5 (gray/black), VE-cadherin (magenta), and DAPI (blue). Cyan box: magnification of RAB5 from highlighted areas, shown below. Scale bar, 10 μm. (G) Quantification of RAB5+ vesicle volume. n = 4 independent experiments; 400–800 vesicles were analyzed per condition for each experiment; mean ± SD; unpaired t test, P = 0.0133 (*). (H)Myct1 ablation enlarges Rab5+ early endosomes in aortic ECs. En face staining of aorta from wild-type or Myct1ecKO mice for Rab5 (gray/black) and VE-cadherin (magenta). Cyan box: magnification of RAB5 from highlighted areas, shown below. Scale bar, 5 μm. (I) Quantification of Rab5+ vesicle volume in aortic ECs. n = 5 mice per genotype; 1,000–4,000 vesicles were analyzed per mouse; mean ± SD; unpaired t test, P = 0.0128 (*). (J)MYCT1 depletion leads to IFITM2/3+ early endosome enlargement. Icons used in J were created with BioRender.com and modified in Affinity. See also Fig. S5, A–G. Source data are available for this figure: SourceData F6.

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