Journal: Journal of diabetes

Article Title: Exercise ameliorating myocardial injury in type 2 diabetic rats by inhibiting excessive mitochondrial fission involving increased irisin expression and AMP-activated protein kinase phosphorylation.

doi: 10.1111/1753-0407.13475

Figure Lengend Snippet: FIGURE 4 Irisin mediates the effect of exercise on mitochondrial fission in diabetic rat hearts involving the phosphorylation of AMPK. (A, B, C) mRNA levels of Drp1, Fis1, and MFF in rat hearts. (D, E, F, G) Protein expression levels of Drp1, Fis1, and MFF in rat hearts and their quantitative analysis. (H, I) Immunofluorescence staining of Drp1 and its quantitative analysis (1000X). (J) Quantitative analysis of AMPK phosphorylation levels in the heart. Data are presented with mean ± SD. * indicates significance (p < .05) compared to the Con group, † presents significance (p < .05) compared to the DM group, and § denotes significance (p < .05) compared to the Ex group. AMPK, AMP-activated protein kinase; Con, control group; Drp1, dynamin-related protein 1; Ex, diabetes plus exercise; ExRg, diabetes plus exercise and Cyclo RGDyk; Fis1, fission-1 protein; MFF, mitochondrial fission factor.

Article Snippet: After blocking PVDF membranes with 5% skimmed milk for 1 h at room temperature, transforming growth factor-β1 (TGF-β1; 1:500, sc-130348, Santa Cruz, USA), irisin (1: 1500, SRP8039, Merck, China), AMPK (1:1000, AF6195, Beyotime, China), P-AMPK (1: 2000, AF5908, Beyotime, China), Drp1 (1:2000, A2586, ABclonal, China), Fis1 (1:2000, CSB-PA197834, Cusabio, China), MFF (1:2000, GB114102, Servicebio, China), and β-actin (1:20000, 66 009-1-lg, Proteintech, China) were detected using the immunoblot at 4 C. The density of protein bands was evaluated with Image-Pro Plus software (version 6.0, Media Cybernetics, USA) and normalized to the equivalent density of the housekeeping protein β-actin.

Techniques: Phospho-proteomics, Expressing, Immunofluorescence, Staining, Control

Journal: Journal of Applied Physiology

Article Title: Role of gp130 in basal and exercise-trained skeletal muscle mitochondrial quality control

doi: 10.1152/japplphysiol.01063.2017

Figure Lengend Snippet: Muscle glycoprotein-130 (gp130) loss effects on mitochondrial biogenesis and content. We next examined mitochondrial biogenesis and content in the gastrocnemius muscle of C57BL/6 mice (B6) and mice with skeletal muscle-specific deletion of gp130 receptor (KO). A: in situ muscle fatigue test of tibialis anterior muscle in B6 (n = 5) and KO (n = 5) mice. B: whole body treadmill run time to fatigue in B6 (n = 5) and KO (n = 5) mice. C: cytochrome-c oxidase (COX) enzymatic activity in crude gastrocnemius homogenate from B6 and KO mice. D: high-succinate dehydrogenase (SDH) activity myofibers expressed as a percentage of total myofibers in B6 and KO mice. E, left: representative immunoblots of phospho (p)-AMPKT172, AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (TFAM), COX IV, and GAPDH in B6 and KO mice. Right: quantification of immunoblots. Max, maximum. Total n = 7 B6 mice and 5 KO mice for C–E. All values are reported as means ± SE. All values were normalized to B6. Analyses were conducted using unpaired Student’s t-test. Statistical significance was set at P < 0.05.

Article Snippet: Primary antibodies for COX I–V, FIS-1 (Abnova), gp130 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-hydroxynonenal (4-HNE; ECM Biosciences), MFN-1, microtubule-associated protein-1 light chain 3B (LC3)-I and LC3-II, Beclin-1, phospho-STAT3 Y705 , STAT3, Total OXPHOS Cocktail (Abcam, Cambridge, United Kingdom), and GAPDH were incubated at 1:2,000 to 1:10,000 dilutions in 5% milk-TBST for 2 h at room temperature or overnight at 4°C.

Techniques: In Situ, Activity Assay, Western Blot

Journal: Journal of Applied Physiology

Article Title: Role of gp130 in basal and exercise-trained skeletal muscle mitochondrial quality control

doi: 10.1152/japplphysiol.01063.2017

Figure Lengend Snippet: Examination of isolated muscle mitochondria. Mitochondria from the gastrocnemius of C57BL/6 mice (B6) and mice with skeletal muscle-specific deletion of gp130 receptor (KO) were isolated and examined for complex protein, mitophagy, and cytochrome-c oxidase (COX) enzymatic activity. A, left: representative immunoblot of total oxidative phosphorylation protein expression in B6 and KO mitochondria. Right: quantification of immunoblot. B, left: representative immunoblots of dynamin-related protein-1 (DRP-1), mitochondrial fission 1 protein (FIS-1), and mitofusin-1 (MFN-1) in B6 and KO mice. Right: quantification of immunoblots. C, left: representative immunoblots of ubiquitin-binding protein p62 (p62), Parkin, and ubiquitin protein in B6 and KO mitochondria. Right: quantification of immunoblots. Dashed line indicates that blot was cropped for representative purposes. D: COX enzymatic activity in B6 and KO mitochondria. CI–V, complexes I–V; mito, mitochondria; VDAC, voltage-dependent anion-selective channel protein. Total n = 6 B6 mice and 6 KO mice. All values are reported as means ± SE. All values were normalized to B6. Analyses were conducted using unpaired Student’s t-test. Statistical significance was set at P < 0.05. *Statistically different from B6.

Article Snippet: Primary antibodies for COX I–V, FIS-1 (Abnova), gp130 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-hydroxynonenal (4-HNE; ECM Biosciences), MFN-1, microtubule-associated protein-1 light chain 3B (LC3)-I and LC3-II, Beclin-1, phospho-STAT3 Y705 , STAT3, Total OXPHOS Cocktail (Abcam, Cambridge, United Kingdom), and GAPDH were incubated at 1:2,000 to 1:10,000 dilutions in 5% milk-TBST for 2 h at room temperature or overnight at 4°C.

Techniques: Isolation, Activity Assay, Western Blot, Phospho-proteomics, Expressing, Ubiquitin Proteomics, Binding Assay

Journal: Journal of Applied Physiology

Article Title: Role of gp130 in basal and exercise-trained skeletal muscle mitochondrial quality control

doi: 10.1152/japplphysiol.01063.2017

Figure Lengend Snippet: Mitochondrial biogenesis and content following exercise training. We next examined the effect of muscle glycoprotein-130 (gp130) loss on gastrocnemius mitochondrial biogenesis and content following 6 wk of treadmill exercise training. A, top: representative immunoblots of gp130, phospho (p)-STAT3Y705, STAT3, and GAPDH in C57BL/6 (B6) exercise-trained (EX) mice. Bottom: quantification of immunoblots above. B, top: representative immunoblots of gp130, p-STAT3Y705, STAT3, and GAPDH in skeletal muscle-specific gp130 knockout (KO) EX mice. Bottom: quantification of immunoblots above. C, left: representative immunoblots of p-AMPKT172, AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (TFAM), cytochrome-c oxidase subunit IV (COX IV), and GAPDH in B6 and KO EX mice. Right: quantification of immunoblots. D: COX enzymatic activity in B6 and KO EX mice. E, top: representative immunoblot of total oxidative phosphorylation protein expression in B6 and KO EX mice. Bottom: quantification of immunoblot above. CC, cage control; CI–V, complexes I–V. Total n = 7 B6 CC mice and 5 KO CC mice, n = 11 B6 EX and 7 KO EX mice. All values were normalized to each genotype’s respective CC. All samples were run on common gels to directly compare samples. Analyses were conducted using two-way ANOVA with Tukey’s post hoc analysis where appropriate. Statistical significance was set at P < 0.05. *Statistically different from CC. #Main effect of KO. &Main effect of exercise.

Article Snippet: Primary antibodies for COX I–V, FIS-1 (Abnova), gp130 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-hydroxynonenal (4-HNE; ECM Biosciences), MFN-1, microtubule-associated protein-1 light chain 3B (LC3)-I and LC3-II, Beclin-1, phospho-STAT3 Y705 , STAT3, Total OXPHOS Cocktail (Abcam, Cambridge, United Kingdom), and GAPDH were incubated at 1:2,000 to 1:10,000 dilutions in 5% milk-TBST for 2 h at room temperature or overnight at 4°C.

Techniques: Western Blot, Knock-Out, Activity Assay, Phospho-proteomics, Expressing, Control

Journal: PLoS ONE

Article Title: Pathological presentation of cardiac mitochondria in a rat model for chronic kidney disease

doi: 10.1371/journal.pone.0198196

Figure Lengend Snippet: Protein expression of the fission protein MFN1 as well as the fusion proteins FIS1 and DRP1. N = 4–6 animals/group. ARB–CKD animals treated with ARB. Data are represented as mean ± SEM.

Article Snippet: [ ] The membranes were incubated with primary antibodies of anti-: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), Angiotensin II type 1 receptor (AT1R), PINK1, GAPDH (Abcam); PARP1 (Santa Cruz); cytosolic cytochrome C (CytC), mitochondrial MFN1, mitochondrial DRP1 (Bios); or with mitochondrial FIS1 (MBL).

Techniques: Expressing