Fig 1: Energy Homeostasis in Adipocyte-Specific Mitofusin 2 Knockout MiceEnergy expenditure analysis (A–D) and respiratory exchange ratio (RER) (E–H) measured over 48 h in Ati-mfn2-CT (n = 8) and Ati-mfn2-KO (n = 8) mice on SD and HFD treatments. Data are mean ± SEM. Statistics: Student’s t test. *p < 0.05, **p < 0.01 versus controls. See also Figure S6.
Fig 2: PSMA7 is required for PARK2-dependent mitophagy-related processes.A Representative western blot images showing PARK6, PARK2 and PSMA7 protein levels in total cell lysate (Input) and MYC-immunoprecipitated lysate (MYC-IP) of HEK 293 T cells transfected with MYC-PARK2 and GFP-PARK6 and either non-targeting control siRNA (siCNT) or PSMA7-targeting siRNA (siP7) and treated with DMSO or CCCP (10 µM) for 12 h. Actin beta (ACTB) was used as loading control. B Graph representing protein levels of PARK6 immunoprecipitated with MYC-PARK2 from A (mean ± SEM, n = 3). C Confocal microscopy analysis of GFP-PARK6 (green) and mCherry-PARK2 (red) expressing HeLa cells co-transfected with siCNT or siP7, and treated with DMSO or CCCP (10 µM) for 12 h. MERGE, overlay of green and red signals. D Quantification of PARK2/PARK6 overlap coefficient of microscopy analysis represented in C (mean ± S.D., n ≥ 30, one-way ANOVA). E and F Representative western blot images of TOM40 (E) and MFN2 (F) levels in MYC-PARK2 expressing HEK 293 T cells co-transfected with siCNT or siP7 and treated with either DMSO or CCCP (10 µM) for 12 h. ACTB was used as loading control. Band intensities were provided below (n = 3). G and H Representative western blot images of ubiquitin levels in TOM40 (G) and MFN2 (H) immunoprecipitated lysates of MYC-PARK2 expressing HEK 293 T cells co-transfected with siCNT or siP7, and treated with DMSO, CCCP (20 µM) or MG132 (30 µM) and CCCP (20 µM) for 2 h. ACTB was used as loading control. Band intensities were provided below (n = 3). I and K, Confocal analyses of MYC-PARK2, GFP-LC3 (green) and mito-dsRed (red) expressing HeLa cells co-transfected with siCNT or siP7, and treated with DMSO or CCCP for 12 h (I) and treated with DMSO, CCCP or O/A for 2 h (K). J and L, Quantification of overlap coeefficiency values of LC3 (green) and mitochondria (red) from I and K respectively (mean ± S.D., n ≥ 40, one-way ANOVA).
Fig 3: Disturbed mitochondrial dynamics in human PMD fibroblasts on fission stimuli. Relative protein expression of DRP1, Fis1, OPA1 and MFN2 in human Control and PLP1‐duplicated PMD fibroblasts (A). Confocal microscopy of stained Control and PMD fibroblasts with anti‐Tom20 antibody and DAPI (nuclei) following exposure to serum depletion for 8 h or CCCP (200 μM) for 1 h (B). Lower panels show enlarged areas of the white boxes in the above panels. Scale bar: 10 uM. Digital images were subjected to a convolve filter through the National Institutes of Health‐developed IMAGEJ software to isolate and equalize fluorescent pixels in the image. After thresholding, individual particles (mitochondria) were analyzed for diameter, average size and circularity. Lower panels show Software processed images (Black and white). A total of 20 cells (n = 3 Ctrl and n = 3 PMD) were examined for each condition. Statistical analysis was performed by one‐way ANOVA and Tukey's HSD post hoc; *P < 0.05, **P < 0.01.
Fig 4: MFN2 Gene Inactivation in Adipose Tissue Leads to Modifications of Mitochondrial Morphology and Overall Adipose Tissue Histology(A) Electron microscopy presentation of epididymal fat from Ati-mfn2-KO mice and controls littermates (Ati-mfn2-CT) both on the SD and HFD. Scale bar, 1 μm.(B–F) Quantification of mitochondria area (in square nanometers; B) and mitochondrial coverage (C) in adipocytes on SD and the same parameters on HFD(D and E) of Ati-mfn2-KO mice and their controls littermates (Ati-mfn2-KO and controls receiving both the SD and HFD, n = 3).(F)Relative change of mitochondrial area on HFD compared to SD values (100%) between Ati-mfn2-CT and Ati-mfn2-KO mice (n = 3 in all groups).(G)Representative H&E staining of epididymal fat from SD- and HFD-fed Ati-mfn2-KO mice and controls. Scale bar, 30 üm.(H and I) Adipocyte cell population distribution according to their cell area SD (H) and HFD (I) of Ati-mfn2-CT and Ati-mfn2-KO mice (n = 3 in all groups).(J) Schematic depicting the time points for BrdU treatment and analysis of adipocyte nuclei by immunofluorescence.(K and L) Quantification of BrdU-positive nuclei in epididymal fat from Ati-mfn2-KO mice and controls receiving either the SD (K) or HFD (L).(M) Quantification of peroxisome proliferator-activated receptor γ (PPAR-γ) mRNA levels in epididymal fat from HFD-treated mice.Data are mean ± SEM. In (F), data from (B)–(D) are presented as variation to control mice receiving the SD. In (K) and (L), data are expressed as percentage variation versus the control group. n = 3 mice for each group, 300–500 cells/group. Statistics: (B)–(E) and (K)–(M), Student’sttest; (F), one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 versus controls. See also Figure S7.
Fig 5: Body Weight, Adipose Tissue Proliferation, and Glucose Homeostasis Are Affected by Adipocyte Deletion of Mitofusin 2(A and B) Body weight analysis of Ati-mfn2-CT and Ati-mfn2-KO mice on the SD (A; controls, n = 16; KO, n = 12) and HFD (B; controls, n = 19; KO, n = 15) at 3, 7, and 12 weeks on the respective diets.(C) Data from (A) and (B) presented as body weight gain (as a percentage) over the 12 weeks of diet treatment.(D and E) MRI of Ati-mfn2-CT and Ati-mfn2-KO mice on SD (D) and HFD (E) correlated the increased body weight of the Ati-mfn2-KO compared with controls because of increased adiposity (the mice from A and B were used for evaluation of body composition by MRI).(F–K) Plasma profile of adiponectin (F), leptin (G), and glucose (H) on standard diet, and adiponectin (I), leptin (J), and glucose (K) on high-fat diet of Ati-mfn2-CT (n = 5–6) and Ati-mfn2-KO (n = 6–7) mice.(L–O) Glucose tolerance test (L and M) and insulin tolerance test (N and O) on Ati-mfn2-CT and Ati-mfn2-KO mice on the SD (controls, n = 5–6; KO, n = 6–8) and HFD (controls, n = 5–6; KO, n = 5–8).Data are mean ± SEM. Statistics: (A), (B), and (D)–(K), Student’s t test; (E), one-way ANOVA; (L)–(O), cumulative quantification of blood glucose in mice during the respective experiments, derived as area under the curve (AUC) and statistically compared by Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S2–S5.
Supplier Page from MilliporeSigma for Anti-Mitofusin 2 antibody produced in rabbit