Journal: Nature communications

Article Title: G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion.

doi: 10.1038/ncomms1099

Figure Lengend Snippet: Figure 4 | G2 requires Mfn1 for its effect on mitochondrial dynamics. (a) Representative images of wild type or Mfn1 − / − and/or Mfn2 − / − MEF cells transfected with Flag-tagged Gβ2. Mitochondria in cells were stained with MitoTracker Red and Flag- Gβ2 were immunostained with a Flag-specific antibody. Scale bar, 10 µm. (b) Mitochondria in Mfn2 − / − cells, and Mfn1 − / − cells are shown in the representative images of Mfn1 − / − or Mfn2 − / − MEFs transfected with Gβ2 shRNA (red). The box area A (tranfected cell) and B (untransfectd cell) in the left panel show the different mitochondrial size at higher magnifications. Scale bar, 10 µm. (c) The quantification of mitochondrial size scored by Image J (Wayne Rasband, NIH). Filled box represents mitochondrial size in Mfn2 − / − MEFs and open box represents in Mfn2 − / − MEFs transfected with Gβ2 shRNA. The data represent the mean s.d., n = 10–15. Differences in the mean mitochondrial average area between mfn2 − / − MEFs and mfn2 − / − MEFs with Gβ2 shRNA were statistically significant at *P < 0.05. (d) Mfn1-YFP was co-transfected into HeLa cells with either Flag- Gβ2 (•, black) or Gβ2 shRNA (, red) and the mitochondrial Mfn1 mobility was measured by FRAP. Fis1 (, green) was used as a control of fast membrane mobility as previously reported28 and Mfn1 alone (, blue) was used as baseline control. Each line represents the mean of ≥20 measurements. (e) The fluorescence recovery of Mfn1-YFP in HeLa cells and the cells co-transfected with either Flag-Gβ2 or Gβ2 shRNA was measured by FRAP. Differences in the mean fluorescence recovery between Mfn1-YFP in HeLa cells and Mfn1-YFP in HeLa cells co-transfected with Flag-Gβ2, or with Gβ2 shRNA were statistically significant *P < 0.05 and **P < 0.005; Error bars represent the s.e.m., n = 21. (f) Mobility of Mfn1-GFP, or Mfn1-GFP (∆241–350), Mfn1-GFP (∆1–387) in HeLa cells and the cells co-transfected with DsRed-Gβ2 shRNA was measured by FRAP. The fluorescence recovery of Mfn1-GFP, Mfn1-GFP (∆241–350) and Mfn1-GFP (∆1–387) in HeLa cells and the cells co-transfected with Gβ2 shRNA was measured by FRAP. *P < 0.05 and NS means nonsignificant. Data represent the mean s.e.m., n = 20.

Article Snippet: Antibodies to Mfn1 and Mfn2 monoclonal were from Novus, the concentrations of both are 500 μg ml − 1.

Techniques: Transfection, Staining, shRNA, Control, Membrane, Fluorescence

Journal: Behavioural brain research

Article Title: Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice.

doi: 10.1016/j.bbr.2024.114889

Figure Lengend Snippet: Fig. 3. RAPA alleviates mitochondrial morphology and functional impairments in 5 ×FAD mice. (A) Representative images of mitochondria morphology in the hippocampal CA1 region under TEM. The red arrowheads indicate mitochondria. Scale bar = 500 nm; (B-D) Quantification of specific surface area (B), numeric density (C), and volume densities of mitochondria (D). n = 3 per group. (E) Relative expressions of proteins related to mitochondrial dynamics determined by western blotting in the cortices homogenate. (F)-(H) Relative quantification analysis of p-Drp1(ser637) (F), MFN2 (G), and OPA1 (H). Oxidative stress injury was evaluated based on the MDA level (I) and SOD activity (J) in the cortex. Energy metabolism was determined based on the ATP level (K) and respiratory chain complex I activity (L) in the cortex. (M) The mitochondrial membrane in the cortex. n = 3 per group.

Article Snippet: The membranes were blocked with QuickblockTM Western blocking buffer (cat. P0252, Beyotime Biotechnology) for 30 min, then subjected to an overnight incubation at 4 ◦C with various primary antibodies at a 1:1000 dilution: β-actin (cat. A2103, Abcam), anti-synapsin (cat. 2312 S, CST) and PSD95 (cat. GB11277, Servicebio), OPA1 (cat. 80741 S, CST) and MFN2 (cat. 11925 S, CST) and DRP1 (cat. 8570 S, CST), Phospho-DRP1(ser637) (cat. 4867 S, CST) and PINK1 (cat. 23274–1-AP, Proteintech) and Parkin (cat. ab77924, Abcam), LC3 (cat. SAB5701328, Sigma), Beclin-1 (cat. 207612, Abcam) and mTOR (cat. 2972 S, CST), and SQSTM1/p62 (cat. ab109012, Abcam).

Techniques: Functional Assay, Western Blot, Quantitative Proteomics, Activity Assay, Membrane

Journal: Behavioural brain research

Article Title: Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice.

doi: 10.1016/j.bbr.2024.114889

Figure Lengend Snippet: Fig. 7. 3-MA counteracts the effect of rapamycin in improving mitochondrial structure and function in 5 ×FAD mice. (A) Representative TEM images of the mitochondrial morphology in the CA1 region. Red arrowheads indicate mitochondria. Scale bar = 500 nm. (B-D) Quantification of the specific surface area (B), numeric density (C), and volume density of mitochondria (D). n = 3 per group. (E) Relative expression of proteins related to mitochondrial dynamics in the cortical homogenate as determined by western blotting. (F)-(H) Relative quantification of p-Drp1(ser637) (F), MFN2 (G), and OPA1 (H). Oxidative stress injury was determined based on MDA level (I) and SOD activity (J), and energy metabolism was determined based on ATP level (K), respiratory chain complex I activity (L), and mitochondrial membrane (M) in the cortices.

Article Snippet: The membranes were blocked with QuickblockTM Western blocking buffer (cat. P0252, Beyotime Biotechnology) for 30 min, then subjected to an overnight incubation at 4 ◦C with various primary antibodies at a 1:1000 dilution: β-actin (cat. A2103, Abcam), anti-synapsin (cat. 2312 S, CST) and PSD95 (cat. GB11277, Servicebio), OPA1 (cat. 80741 S, CST) and MFN2 (cat. 11925 S, CST) and DRP1 (cat. 8570 S, CST), Phospho-DRP1(ser637) (cat. 4867 S, CST) and PINK1 (cat. 23274–1-AP, Proteintech) and Parkin (cat. ab77924, Abcam), LC3 (cat. SAB5701328, Sigma), Beclin-1 (cat. 207612, Abcam) and mTOR (cat. 2972 S, CST), and SQSTM1/p62 (cat. ab109012, Abcam).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Activity Assay, Membrane