Journal: Nutrients

Article Title: β-Cryptoxanthin Improves p62 Accumulation and Muscle Atrophy in the Soleus Muscle of Senescence-Accelerated Mouse-Prone 1 Mice

doi: 10.3390/nu12082180

Figure Lengend Snippet: Repression of muscle atrophy by β-cryptoxanthin in the soleus muscle of SAMP1 mice. ( A ) Immunofluorescent staining of laminin in the soleus muscle of the SAMR1 (R1), SAMP1-control (P1con), and SAMP1-CX (P1cx) groups. Scale bar: 100 μm. ( B ) Distribution of soleus muscle fiber cross-sectional area (CSA). ( C ) Average size of the soleus muscle fiber CSA. ( D ) Western blot analyses of MyHC type I and GAPDH. MyHC type I expression was normalized to anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Values are represented as mean ± standard deviation. n = 5–8 per group. * p < 0.05, statistical significance compared with the SAMP1-control group.

Article Snippet: Tissue homogenates and cell lysates were centrifuged at 20,000× g for 15 min, and the supernatants were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by Western blot analysis using the following antibodies: rabbit polyclonal beclin-1 (cell signaling; Danvers, MA, USA), anti-p62, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [ ], and anti-ubiquitin (cell signaling) antibodies; rabbit monoclonal anti-LC3 (clone D3U4C; cell signaling), anti-atrogin-1 (clone EPR9148(2); Abcam; Cambridge, UK), anti-p70S6K (clone 49D7; cell signaling), anti-phospho-p70S6K (clone 108D2; cell signaling), anti-AMPKα (clone 40H9; cell signaling), anti-mTOR (clone 7C10; cell signaling), and anti-phospho-mTOR (clone D9C2; cell signaling) antibodies; mouse monoclonal anti-β-actin (clone 2D4H5; cell signaling) and anti-myosin heavy chain (MyHC) (clone BA-D5 that recognizes MyHC type I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) antibodies; and goat polyclonal anti-MuRF-1 antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Control, Western Blot, Expressing, Standard Deviation

Journal: Scientific Reports

Article Title: Loss of microRNA-23–27–24 clusters in skeletal muscle is not influential in skeletal muscle development and exercise-induced muscle adaptation

doi: 10.1038/s41598-018-37765-3

Figure Lengend Snippet: Skeletal muscle phenotype of miR-23–27–24-cluster-dKO mice. ( A ) Representative images of western blotting analysis of plantaris muscle. ( B ) Quantitative data for the protein expression of MyHC type IIa, MyHC type IIb, PGC-1α, and COX IV. Data are means ± SEM (n = 6). ( C ) Representative images of western blotting analysis of soleus muscle. ( D ) Quantitative data for the protein expression of MyHC type I, MyHC type IIa, PGC-1α, and COX IV. Data are means ± SEM (n = 5–6). ( E ) Representative images of plantaris and soleus muscles immunofluorescently stained for MyHC type IIa fibers and dystrophin in green, and type IIb fibers in red. MyHC type IId/x fibers are unstained (black). Scale bar, 50 μm. ( F ) Proportions of muscle fiber types in plantaris and soleus muscles. Proportion of each fiber type is presented as a percentage. Muscle fibers in entire cross-sections were counted. I, MyHC type I; IIa, MyHC type IIa; IIb, MyHC type IIb; IId/x, MyHC type IId/x. Data are means ± SEM (n = 4–5). # P < 0.05 compared with WT. ( G ) Total fiber numbers in plantaris and soleus muscles. Muscle fibers in entire cross-sections were counted. Data are means ± SEM (n = 4–5). ( H ) Distributions of MyHC type IIa fibers (top) MyHC type IIb fibers (middle), and MyHC type IId/x fibers (bottom) in plantaris muscle. Graphs show percentages of fibers per cross-sectional area (CSA). Data are means ± SEM (n = 5). ( I ) CSA distributions of MyHC type I fibers (top), MyHC type IIa (middle), and MyHC type IId/x fibers (bottom) in soleus muscle. Data are means ± SEM (n = 5). ( J ) mRNA expression of Myostatin, MurF1 and Atrogin-1 in WT and dKO mice skeletal muscle. Data are means ± SEM (n = 6). K, Quantitative data for the phospho-Akt/Akt by western blotting in WT and dKO mice skeletal muscle. Data are means ± SEM (n = 6).

Article Snippet: The membranes were incubated with the following primary antibodies: anti-myosin heavy chain (MyHC) type I (BA-F8, DSMZ, 1:100), anti-MyHC type IIa (SC-71, DSMZ, 1:100), anti-MyHC type IIb (BF-F3, DSMZ, 1:100), anti-PGC1-α (AB3242, Millipore, 1:500), anti-cytochrome c oxidase complex IV (anti-COX IV; #4844, Cell Signaling Technology, 1:1000), anti-CD31 (anti-PECAM-1; sc-46694, Santa Cruz Biotechnology, INC., 1:400), anti-Phospho-Akt (#4060, Cell signaling Technology, 1:1000), anti-Akt (#9272, Cell signaling Technology, 1:1000) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; MAB374, Millipore, 1:1000).

Techniques: Western Blot, Expressing, Muscles, Staining

Journal: Scientific Reports

Article Title: Loss of microRNA-23–27–24 clusters in skeletal muscle is not influential in skeletal muscle development and exercise-induced muscle adaptation

doi: 10.1038/s41598-018-37765-3

Figure Lengend Snippet: miR-23–27–24 clusters are not essential for endurance-exercise-induced muscle adaptation. ( A ) Representative images of western blotting analysis. ( B ) Quantitative data for the protein expression of MyHC type IIa, MyHC type IIb, PGC-1α, and COX IV. Data are means ± SEM (n = 6). **P < 0.01 main effect of exercise. ( C ) Representative images of SDH staining in plantaris muscle. Scale bar, 100 μm. ( D ) Representative immunofluorescent images of capillaries in plantaris muscle. Dystrophin is shown in red, CD31 in green. Scale bar, 100 μm. Quantitative data for the capillary-to-fiber ratio ( E ) and CD31 expression ( F ) in plantaris muscle. Data are means ± SEM (n = 5). **P < 0.01 main effect of exercise. ( G ) Representative images of plantaris muscle immunofluorescently stained for MyHC type IIa fibers and dystrophin in green, type IIb fibers in red. MyHC type IId/x fibers are unstained (black). Scale bar, 500 μm. ( H ) Mean cross-sectional areas (CSAs) of MyHC type IIa, MyHC type Id/x, and MyHC type IIb fibers in plantaris muscle. Data are means ± SEM (n = 4–5). # P < 0.05 main effect of genotype. **P < 0.01 main effect of exercise. ( I ) Proportions of MyHC IIa, MyHC IId/x, and MyHC IIb fibers in plantaris muscle. Data are means ± SEM (n = 4). # P < 0.05 main effect of genotype. **P < 0.01 main effect of exercise. ( J ) Expressions of the mature miRNAs in miR-23a/b clusters after 4 weeks of voluntary running. Data are means ± SEM (n = 5). ## P < 0.01 main effect of genotype. *P < 0.05 main effect of exercise. sed, sedentary group; ex, exercised group.

Article Snippet: The membranes were incubated with the following primary antibodies: anti-myosin heavy chain (MyHC) type I (BA-F8, DSMZ, 1:100), anti-MyHC type IIa (SC-71, DSMZ, 1:100), anti-MyHC type IIb (BF-F3, DSMZ, 1:100), anti-PGC1-α (AB3242, Millipore, 1:500), anti-cytochrome c oxidase complex IV (anti-COX IV; #4844, Cell Signaling Technology, 1:1000), anti-CD31 (anti-PECAM-1; sc-46694, Santa Cruz Biotechnology, INC., 1:400), anti-Phospho-Akt (#4060, Cell signaling Technology, 1:1000), anti-Akt (#9272, Cell signaling Technology, 1:1000) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; MAB374, Millipore, 1:1000).

Techniques: Western Blot, Expressing, Staining