Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF-β2/SMAD Signaling Pathway

doi: 10.3390/ijms21093274

Figure Lengend Snippet: MiR-200a-3p directly targets TGF-β2 in skeletal muscle satellite cells (SMSCs). ( a ) Prediction of target genes using TargetScan, miRDB, and Diana. ( b ) Seed region of miR-200a-3p. ( c ) Dual-luciferase reporter gene (pEZX-FR02) with wild type (pEZX- TGF-β2 -WT) or mutant (pEZX- TGF-β2 -MT). ( d ) Luciferase assays were performed by cotransfection of wild type or mutant TGF-β2 3′ UTR with a miR-200a-3p mimic or mimic-NC in SMSCs. ( e , f ) After transfection of miR-200a-3p mimics, miR-200a-3p inhibitors or NC, the expression of TGF-β2 was determined by q-PCR and Western blot. Data are expressed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: The antibodies used for the experiments were as follows: Anti-MyHC (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Abcam, London, UK; 1:1,000 dilution),anti-caspase-9 (Abcam, London, UK; 1:1000 dilution), anti-TGF-β2 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution), anti-β-actin (Santa Cruz Biotechnology; 1:1000 dilution), anti-SMAD2/3 and anti-p-SMAD2/3 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution). β-actin was used as the loading control.

Techniques: Luciferase, Mutagenesis, Cotransfection, Transfection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF-β2/SMAD Signaling Pathway

doi: 10.3390/ijms21093274

Figure Lengend Snippet: Effects of TGF-β2 on differentiation of skeletal muscle satellite cells (SMSCs). ( a , b ) After overexpression or interference with TGF-β2, qRT-PCR was used to detect the expression of TGF-β2 in SMSCs. ( c , d ) The relative expressions of MyoD, MyHC, and MyoG mRNA in SMSCs differentiated for 48 h. ( e ) Western blot detects protein levels of myogenic marker genes after inhibition and overexpression of TGF-β2. ( f ) Representative images of immunofluorescent staining of differentiated SMSCs (200×). Myosin: Red, a molecular marker of myogenesis; DAPI: Blue, cell nuclei; Merge: The fusion of SMSCs into primary myotubes. ( g ) Myotube area (%) and MYHC positive cells (%) after transfection of si-TGF-β2, pcDNA3.1-TGF-β2, or NC. Data are expressed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01 vs. NC.

Article Snippet: The antibodies used for the experiments were as follows: Anti-MyHC (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Abcam, London, UK; 1:1,000 dilution),anti-caspase-9 (Abcam, London, UK; 1:1000 dilution), anti-TGF-β2 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution), anti-β-actin (Santa Cruz Biotechnology; 1:1000 dilution), anti-SMAD2/3 and anti-p-SMAD2/3 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution). β-actin was used as the loading control.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Marker, Inhibition, Staining, Transfection

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF-β2/SMAD Signaling Pathway

doi: 10.3390/ijms21093274

Figure Lengend Snippet: Effects of TGF-β2 on apoptosis of skeletal muscle satellite cells (SMSCs). ( a , b ) Scattergram and the rate of apoptosis in SMSCs transfected with promoting or inhibiting TGF-β2 expression as analyzed using flow cytometry following staining with annexin V and PI. ( c , d ) Abundance of mRNA and proteins of caspase-3 and caspase-9 in SMSCs transfected by the use of qPCR and Western blot analysis. Data are expressed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01 vs. NC.

Article Snippet: The antibodies used for the experiments were as follows: Anti-MyHC (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Abcam, London, UK; 1:1,000 dilution),anti-caspase-9 (Abcam, London, UK; 1:1000 dilution), anti-TGF-β2 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution), anti-β-actin (Santa Cruz Biotechnology; 1:1000 dilution), anti-SMAD2/3 and anti-p-SMAD2/3 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution). β-actin was used as the loading control.

Techniques: Transfection, Expressing, Flow Cytometry, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF-β2/SMAD Signaling Pathway

doi: 10.3390/ijms21093274

Figure Lengend Snippet: MiR-200a-3p regulates differentiation and apoptosis of skeletal muscle satellite cells (SMSCs) via the TGF-β/SMAD pathway. ( a ) After overexpression of TGF-β2/TGF-β2 + miR-200a-3p/TGF-β2 + ITD-1, the protein levels of TGF-β/SMAD pathway genes were detected using Western blotting. ( b , c ) Effect of miR-200a-3p mimics on differentiation and apoptosis of SMSC cells in the absence or presence of ITD-1. Data are expressed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01 vs. NC.

Article Snippet: The antibodies used for the experiments were as follows: Anti-MyHC (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Abcam, London, UK; 1:1,000 dilution),anti-caspase-9 (Abcam, London, UK; 1:1000 dilution), anti-TGF-β2 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution), anti-β-actin (Santa Cruz Biotechnology; 1:1000 dilution), anti-SMAD2/3 and anti-p-SMAD2/3 (ABclonal, Wuhan, Hubei, China; 1:1000 dilution). β-actin was used as the loading control.

Techniques: Over Expression, Western Blot