Figure 2.
Reduced adiposity in Myct1ecKOmice is independent of angiogenesis, adipogenesis, and systemic metabolic activity. (A) Experimental workflow for monitoring early changes in metabolism of Myct1ecKO mice. (B) Food intake over 24 h (23°C) is similar between wild-type and Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 CD45negCD31+ 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 Myct1ecKO 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 Myct1ecKO 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 CD45negCD31negSca1+ 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 Myct1ecKO 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 Fig. S2. Refer to the image caption for details. Panel A shows an experimental workflow diagram illustrating tamoxifen induction in mice followed by metabolic monitoring using CLAMS and Echo M R I before analysis. Panel B is a bar graph comparing food intake over 24 hours between wildtype and M y c t 1 e c K O mice, indicating similar intake levels. Panel C is a bar graph showing total horizontal activity over 24 hours, also similar between the two groups. Panel D is a bar graph of the respiratory exchange ratio over 24 hours, showing no significant difference. Panel E is a bar graph of energy expenditure over 24 hours, again similar between wildtype and M y c t 1 e c K O mice. Panel F shows Masson's trichrome staining images of retroperitoneal fat sections, with Panel G quantifying fibrosis as a percentage of area positive for collagen. Panel H displays staining of retroperitoneal sections for Pecam 1 and Fabp 4, with Panel I quantifying vascular density. Panel J quantifies C D 4 5 n e g C D 31 plus cells as a percentage of total cells. Panel K outlines the experimental workflow for analyzing postnatal retina angiogenesis. Panel L shows staining of P 5 and P 7 retina for Pecam1, with Panel M quantifying vascular outgrowth and Panel N quantifying vascular density. Panel O quantifies C D 45 plus immune cells, Panel P quantifies C D 45 n e g C D 31 n e g S c a 1 plus adipocyte progenitor cells, and Panel Q shows the experimental workflow for an ex vivo adipogenesis assay. Panel R displays staining of stromal vascular fraction for Perilipin and D N A, with Panel S quantifying Perilipin plus area normalized to cell number. Panel T illustrates the effect of M y c t 1 ablation on white adipose tissue expansion.

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 Myct1ecKO mice. (B) Food intake over 24 h (23°C) is similar between wild-type and Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 Myct1ecKO 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 CD45negCD31+ 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 Myct1ecKO 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 Myct1ecKO 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 CD45negCD31negSca1+ 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 Myct1ecKO 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 Fig. S2.

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