Journal: Cell Reports Medicine

Article Title: A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs

doi: 10.1016/j.xcrm.2021.100298

Figure Lengend Snippet:

Article Snippet: Western blot analysis was performed using the iBind Flex Western Device (Thermo Fisher), PVDF membranes (Bio-Rad), and the following primary antibodies: mouse anti-myosin heavy chain (pan-MHC) monoclonal antibody (MF20; 1:1,000, R&D), mouse anti-skeletal myosin (FAST/MYH1&2) monoclonal antibody (MY-32; 1:800, Sigma), mouse anti-Myosin-2 (MYH2) monoclonal antibody (MABT840; 1:1,000, Millipore), rabbit anti-MYH3 polyclonal antibody (1:1000, Atlas antibodies), mouse anti-MYH7 monoclonal antibody (A4.840; 1:500, Santa Cruz), rabbit anti-MYH8 polyclonal antibody (1:1,000, Novus Biologicals), rabbit anti-RYR1 monoclonal antibody (D4E1; 1:500, Cell Signaling Technology, Danvers, MA), mouse anti-myogenin monoclonal antibody (F5D; 1:200, Santa Cruz), mouse anti-dystrophin (Rod domain) monoclonal antibody (DYS1; 1:20, Leica), rabbit anti-TBP monoclonal antibody (D5C9H; 1:1000, Cell Signaling).

Techniques: Recombinant, Knock-Out, Blocking Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Reports Medicine

Article Title: A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs

doi: 10.1016/j.xcrm.2021.100298

Figure Lengend Snippet:

Article Snippet: Western blot analysis was performed using the iBind Flex Western Device (Thermo Fisher), PVDF membranes (Bio-Rad), and the following primary antibodies: mouse anti-myosin heavy chain (pan-MHC) monoclonal antibody (MF20; 1:1,000, R&D), mouse anti-skeletal myosin (FAST/MYH1&2) monoclonal antibody (MY-32; 1:800, Sigma), mouse anti-Myosin-2 (MYH2) monoclonal antibody (MABT840; 1:1,000, Millipore), rabbit anti-MYH3 polyclonal antibody (1:1000, Atlas antibodies), mouse anti-MYH7 monoclonal antibody (A4.840; 1:500, Santa Cruz), rabbit anti-MYH8 polyclonal antibody (1:1,000, Novus Biologicals), rabbit anti-RYR1 monoclonal antibody (D4E1; 1:500, Cell Signaling Technology, Danvers, MA), mouse anti-myogenin monoclonal antibody (F5D; 1:200, Santa Cruz), mouse anti-dystrophin (Rod domain) monoclonal antibody (DYS1; 1:20, Leica), rabbit anti-TBP monoclonal antibody (D5C9H; 1:1000, Cell Signaling).

Techniques: Recombinant, Knock-Out, Blocking Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: The Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice

doi: 10.3390/ijms25042148

Figure Lengend Snippet: Comparison of the expression levels of sarcoplasmic reticulum calcium ion ATPase-related factors in skeletal muscle. Relative mRNA expression level of Atp2a1 ( a , b ), Atp2a2 ( c , d ), Sln ( e , f ), and Pln ( g , h ) in the gastrocnemius and soleus muscles. Data are presented as mean ± standard deviation, n = 7–8 per group. Significant differences between young and old mice were determined using Student’s t -test. ** p < 0.001, * p < 0.05. n.s.: not significant. All data are represented as dots in the graph. GAS, gastrocnemius muscle; SOL, soleus muscle; 3M, 3-month-old mice; 24M, 24-month-old mice.

Article Snippet: The sections were then bleached with 3% H 2 O 2 , rinsed with PBS, and incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 at 4 °C for 1 h. The sections were then incubated with mouse monoclonal anti-DHPRα1 antibodies (MA3-920; Thermo Fisher Scientific, Waltham, MA, USA; 1:100) or rabbit polyclonal anti-SERCA1 antibodies (22361-1-AP; Proteintech, Rosemont, IL, USA; 1:500), anti-SERCA2 antibodies (ab3625; Abcam, Cambridge, MA, USA; 1:500), anti-RyR1 antibodies (D4E1, #8153; Cell Signaling Technology, Danvers, MA, USA; 1:100), anti-JPH1 antibodies (40-5100; Thermo Fisher Scientific, Waltham, MA, USA; 1:250), anti-JPH2 antibodies (40-5300; Thermo Fisher Scientific, Waltham, MA, USA; 1:50), or anti-MMP2 antibodies (GTX104577; GeneTex, Irvine, CA, USA; 1:50) in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. Subsequently, the sections were incubated with biotinylated anti-mouse or rabbit immunoglobulin G (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) diluted at 1:1000 in PBS for 1 h at 25 °C, followed by incubation with avidin-biotin complex (Vectastain ABC kit) for 1 h at 4 °C.

Techniques: Comparison, Expressing, Muscles, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice

doi: 10.3390/ijms25042148

Figure Lengend Snippet: Sarcoplasmic reticulum calcium ion ATPase (SERCA) localization. Cross-sections of the gastrocnemius ( a , c , e , g ) and soleus ( b , d , f , h ) muscles were stained with anti-SERCA1 ( a – d ) or anti-SERCA2a ( e – h ) antibodies. Asterisks indicate normal immunoreactivity (IR) of SERCA1 or SERCA2a in the cytoplasm ( a , b , d – f , h ). Arrows indicate muscle fiber with ectopic IR of SERCA1 or SERCA2a ( c , g ). Scale bar = 50 μm. The number of muscle fibers with ectopic SERCA1 or SERCA2a IR was measured in the gastrocnemius ( i , k ) and soleus ( j , l ) muscles. Data are presented as mean ± standard deviation, n = 8 per group. Significant differences between young and old mice were determined using Student’s t -test. *** p < 0.0001. n.s.: not significant. All data are represented as dots in the graph. GAS, gastrocnemius muscle; SOL, soleus muscle; 3M, 3-month-old mice; 24M, 24-month-old mice.

Article Snippet: The sections were then bleached with 3% H 2 O 2 , rinsed with PBS, and incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 at 4 °C for 1 h. The sections were then incubated with mouse monoclonal anti-DHPRα1 antibodies (MA3-920; Thermo Fisher Scientific, Waltham, MA, USA; 1:100) or rabbit polyclonal anti-SERCA1 antibodies (22361-1-AP; Proteintech, Rosemont, IL, USA; 1:500), anti-SERCA2 antibodies (ab3625; Abcam, Cambridge, MA, USA; 1:500), anti-RyR1 antibodies (D4E1, #8153; Cell Signaling Technology, Danvers, MA, USA; 1:100), anti-JPH1 antibodies (40-5100; Thermo Fisher Scientific, Waltham, MA, USA; 1:250), anti-JPH2 antibodies (40-5300; Thermo Fisher Scientific, Waltham, MA, USA; 1:50), or anti-MMP2 antibodies (GTX104577; GeneTex, Irvine, CA, USA; 1:50) in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. Subsequently, the sections were incubated with biotinylated anti-mouse or rabbit immunoglobulin G (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) diluted at 1:1000 in PBS for 1 h at 25 °C, followed by incubation with avidin-biotin complex (Vectastain ABC kit) for 1 h at 4 °C.

Techniques: Muscles, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice

doi: 10.3390/ijms25042148

Figure Lengend Snippet: Histochemical findings and ectopic sarcoplasmic reticulum calcium ion ATPase (SERCA) immunoreactivity (IR). Serial cross-sections of the gastrocnemius muscles of old ( a – c ) and young mice ( f – h ) were stained with hematoxylin and eosin (HE), modified Gomori’s trichrome, and succinate dehydrogenase (SDH). These cross-sections were also stained with anti-SERCA1 or anti-SERCA2a antibodies in old ( d , e ) and young mice ( i , j ). Black arrowheads indicate spikes and lakes in the cytoplasm ( a ). White arrowheads indicate red materials in cytoplasm ( b ). Black arrows indicate ectopic SERCA IR ( d , e ). Asterisks denote identical muscle fibers. Scale bar = 50 μm. GAS, gastrocnemius muscle; 3M, 3-month-old mice; 24M, 24-month-old mice.

Article Snippet: The sections were then bleached with 3% H 2 O 2 , rinsed with PBS, and incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 at 4 °C for 1 h. The sections were then incubated with mouse monoclonal anti-DHPRα1 antibodies (MA3-920; Thermo Fisher Scientific, Waltham, MA, USA; 1:100) or rabbit polyclonal anti-SERCA1 antibodies (22361-1-AP; Proteintech, Rosemont, IL, USA; 1:500), anti-SERCA2 antibodies (ab3625; Abcam, Cambridge, MA, USA; 1:500), anti-RyR1 antibodies (D4E1, #8153; Cell Signaling Technology, Danvers, MA, USA; 1:100), anti-JPH1 antibodies (40-5100; Thermo Fisher Scientific, Waltham, MA, USA; 1:250), anti-JPH2 antibodies (40-5300; Thermo Fisher Scientific, Waltham, MA, USA; 1:50), or anti-MMP2 antibodies (GTX104577; GeneTex, Irvine, CA, USA; 1:50) in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. Subsequently, the sections were incubated with biotinylated anti-mouse or rabbit immunoglobulin G (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) diluted at 1:1000 in PBS for 1 h at 25 °C, followed by incubation with avidin-biotin complex (Vectastain ABC kit) for 1 h at 4 °C.

Techniques: Muscles, Staining, Modification

Journal: International Journal of Molecular Sciences

Article Title: The Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice

doi: 10.3390/ijms25042148

Figure Lengend Snippet: DHPRα1 and SERCA1 co-localization. Cross sections of the old gastrocnemius muscles were stained by anti-DHPRα1 ( a ) and SERCA1 ( b ) antibodies. DAPI was used for counterstaining ( c ). Arrowheads indicate representative localizations of DHPRa1 ( a ) and SERCA1 ( b ) in tubular aggregates (TAs), and arrowheads of overlay image shows representative co-localizations of DHPRa1 and SERCA1 in TAs ( d ). Scale bar = 10 μm. GAS, gastrocnemius muscle; 24M, 24-month-old mice.

Article Snippet: The sections were then bleached with 3% H 2 O 2 , rinsed with PBS, and incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 at 4 °C for 1 h. The sections were then incubated with mouse monoclonal anti-DHPRα1 antibodies (MA3-920; Thermo Fisher Scientific, Waltham, MA, USA; 1:100) or rabbit polyclonal anti-SERCA1 antibodies (22361-1-AP; Proteintech, Rosemont, IL, USA; 1:500), anti-SERCA2 antibodies (ab3625; Abcam, Cambridge, MA, USA; 1:500), anti-RyR1 antibodies (D4E1, #8153; Cell Signaling Technology, Danvers, MA, USA; 1:100), anti-JPH1 antibodies (40-5100; Thermo Fisher Scientific, Waltham, MA, USA; 1:250), anti-JPH2 antibodies (40-5300; Thermo Fisher Scientific, Waltham, MA, USA; 1:50), or anti-MMP2 antibodies (GTX104577; GeneTex, Irvine, CA, USA; 1:50) in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. Subsequently, the sections were incubated with biotinylated anti-mouse or rabbit immunoglobulin G (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) diluted at 1:1000 in PBS for 1 h at 25 °C, followed by incubation with avidin-biotin complex (Vectastain ABC kit) for 1 h at 4 °C.

Techniques: Muscles, Staining