Journal: Journal of animal science

Article Title: MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

doi: 10.1093/jas/skac238

Figure Lengend Snippet: Figure 2. MiR-18 inhibitor promotes bMDSCs differentiation. (A, C). Immunofluorescence staining of Desmin (FITC) in DM or GM. bMDSCs were transfected with miR-18 inhibitors, NC (negative control; scrambled sequence), or miR-17 and miR-19 (positive control) in DM or GM for 3, 5, and 7 d. Scale bars, 100 µm. (B, D). Muscular tube fusion rate in accordance with the Desmin staining in A and C. *P < 0.05, **P < 0.01, and ***P < 0.001. (E, F). Western blotting of MYH3 expression from bMDSCs in DM or GM processed with miR-18 inhibitor, NC or miR-17 and miR-19 for 48 h. GAPDH was employed to serve as a loading control.

Article Snippet: We used primary antibodies for Anti-MYH3 (1:1000, cat# 22287-1-AP), Anti-FLAG (1:1000, cat# 66008-4-Ig), and Anti-GAPDH (1:1000, cat# 10494-1-AP) were purchased from Proteintech (Chicago, IL) for western blot analysis.

Techniques: Immunofluorescence, Staining, Transfection, Negative Control, Sequencing, Positive Control, Western Blot, Expressing, Control

Journal: Journal of animal science

Article Title: MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

doi: 10.1093/jas/skac238

Figure Lengend Snippet: Figure 4. MiR-18 inhibitor promotes differentiation of bMDSCs through targeted inhibition of MEF2D expression. (A) MEF2D overexpression was confirmed by western blotting of the Flag tag in transfected bMDSCs. GAPDH was employed to serve as a loading control. ***P < 0.001. (B) Desmin immunostaining of bMDSCs transfected with MEF2D or control vectors and cultured in DM or GM medium on day 3. Scale bars, 100 µm. (C) Muscular tube fusion rates in B in accordance with Desmin staining. ***P < 0.001. (D) MYH3 expression in bMDSCs that overexpressed MEF2D in DM or GM for 48 h. **P < 0.01. (E) Desmin immunostaining of bMDSCs transfected with miR-18 inhibitor together with MEF2D or miR-18 mimics combined with MEF2D. Scale bars, 100 µm. F. Muscular tube fusion rates in E in accordance with the Desmin staining. *P < 0.05.

Article Snippet: We used primary antibodies for Anti-MYH3 (1:1000, cat# 22287-1-AP), Anti-FLAG (1:1000, cat# 66008-4-Ig), and Anti-GAPDH (1:1000, cat# 10494-1-AP) were purchased from Proteintech (Chicago, IL) for western blot analysis.

Techniques: Inhibition, Expressing, Over Expression, Western Blot, FLAG-tag, Transfection, Control, Immunostaining, Cell Culture, Staining

Journal: Journal of animal science

Article Title: MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

doi: 10.1093/jas/skac238

Figure Lengend Snippet: Figure 5. Combined miR-19 and miR-18 inhibitor significantly improve the differentiation of bMDSCs. (A) Desmin immunofluorescence (FITC) was used to detect the differentiation of bMDSCs. Cells were transfected with miR-17 mimic, miR-18 inhibitor, miR-19 mimic, miR-18 inhibitor, miR-17 mimic, miR-19, or NC. Scale bars, 100 µm. (B) Muscular tube fusion rates in A in accordance with Desmin staining. *P < 0.01 and ***P < 0.001. (C) The relative amounts of MYH3 and MYOG mRNAs were detected using qRT-PCR, after bMDSCs were transfected with miR-17 mimic and miR-18 inhibitor, miR-19 mimic and miR-18 inhibitor, miR-17 mimic and miR-19 mimic, or NC. ***P < 0.001. (D) Western blotting to detect MYH3 expression in bMDSCs treated with miR-17 mimics and miR-18 inhibitor, miR-19 mimics and miR-18 inhibitor, miR-17 mimic and miR-19 mimic or NC for 72 h. GAPDH was used as a loading control.

Article Snippet: We used primary antibodies for Anti-MYH3 (1:1000, cat# 22287-1-AP), Anti-FLAG (1:1000, cat# 66008-4-Ig), and Anti-GAPDH (1:1000, cat# 10494-1-AP) were purchased from Proteintech (Chicago, IL) for western blot analysis.

Techniques: Immunofluorescence, Transfection, Staining, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Annals of Translational Medicine

Article Title: MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats

doi: 10.21037/atm-20-8212

Figure Lengend Snippet: The primers used in qRT-PCR

Article Snippet: The antibodies used in this part were as following, CD90 (Invitrogen Catalog #MA1-80650); CD44 (Invitrogen Catalog #MA5-17520); CD45 (Invitrogen Catalog #12-0461-82); Cav1 (Bioss,bs-1453R); MyHC (proteintech, 22281-1-AP); Goat anti-rabbit cy3 (proteintech SA00009-2); GAPDH (proteintech, 60004-1-Ig); MyoD (proteintech, 18943-1-AP); Collagen III (Bioss, bs-0549R); Collagen I (abcam, ab260043); LAMC1 (cell signaling, #92921); MMP1 (Affinify, DF6325); MMP9 (abcam, ab76003); HOXA11 (Bioss, bs-666R); Elastin (Bioss, bs-1756R); Calponin (abcam, ab46794); Vimentin (abcam, ab92547); GFP (abcam, ab1218).

Techniques: Sequencing

Journal: Annals of Translational Medicine

Article Title: MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats

doi: 10.21037/atm-20-8212

Figure Lengend Snippet: MiR-124-3p over-expression accelerates the myogenic differentiation of ADSCs. The expression of miR-124-3p (A), Cav1 (B), Myod (C), and MyHC (D) were tested by qRT-PCR. The expression of Cav1 (E) and MyHC (F) were detected by immunofluorescence. ADSCs, adipose-derived stem cells. *P<0.05, **P<0.01 vs. ADSCs-GFP group.

Article Snippet: The antibodies used in this part were as following, CD90 (Invitrogen Catalog #MA1-80650); CD44 (Invitrogen Catalog #MA5-17520); CD45 (Invitrogen Catalog #12-0461-82); Cav1 (Bioss,bs-1453R); MyHC (proteintech, 22281-1-AP); Goat anti-rabbit cy3 (proteintech SA00009-2); GAPDH (proteintech, 60004-1-Ig); MyoD (proteintech, 18943-1-AP); Collagen III (Bioss, bs-0549R); Collagen I (abcam, ab260043); LAMC1 (cell signaling, #92921); MMP1 (Affinify, DF6325); MMP9 (abcam, ab76003); HOXA11 (Bioss, bs-666R); Elastin (Bioss, bs-1756R); Calponin (abcam, ab46794); Vimentin (abcam, ab92547); GFP (abcam, ab1218).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Immunofluorescence, Derivative Assay

Journal: Annals of Translational Medicine

Article Title: MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats

doi: 10.21037/atm-20-8212

Figure Lengend Snippet: Cav1 is a target of miR-124-3p. (A) The potential binding sites between miR-124-3p and Cav1. (B) Luciferase reporter activity in ADSCs transfected with WT, MUT 3'-UTR of Cav1. **P<0.01 vs. mimic NC. (C) qRT-RCR reveals mRNA expression levels of Cav1, Myod, Myog, and MyHC in ADSCs. (D) Western blot reveals the protein levels of Cav1, Myod, Myog, and MyHC in ADSCs. (E) The expression of MyHC was detected by immunofluorescence. (C-E) *P<0.05, **P<0.01 vs. ADSC-GFP; #P<0.05, ##P<0.01 vs miR-124-3p mimic NC; &&P<0.01 vs. empty vector. qRT-PCR, real-time quantitative polymerase chain reaction; ADSCs, adipose-derived stem cells.

Article Snippet: The antibodies used in this part were as following, CD90 (Invitrogen Catalog #MA1-80650); CD44 (Invitrogen Catalog #MA5-17520); CD45 (Invitrogen Catalog #12-0461-82); Cav1 (Bioss,bs-1453R); MyHC (proteintech, 22281-1-AP); Goat anti-rabbit cy3 (proteintech SA00009-2); GAPDH (proteintech, 60004-1-Ig); MyoD (proteintech, 18943-1-AP); Collagen III (Bioss, bs-0549R); Collagen I (abcam, ab260043); LAMC1 (cell signaling, #92921); MMP1 (Affinify, DF6325); MMP9 (abcam, ab76003); HOXA11 (Bioss, bs-666R); Elastin (Bioss, bs-1756R); Calponin (abcam, ab46794); Vimentin (abcam, ab92547); GFP (abcam, ab1218).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Western Blot, Immunofluorescence, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Multi-Omics Insights into Postnatal Skeletal Muscle Development in Duroc Pigs

doi: 10.3390/ani15182715

Figure Lengend Snippet: Phenotypic assessment of SOL, GAS, and PMM in Duroc pigs. ( A ) Experimental design diagram. ( B ) Phenotypic diagrams of SOL, GAS, LDM, and PMM are presented, with LDM as the control for PMM ( n = 6). ( C ) HE staining of SOL, GAS, and PMM across three developmental stages ( n = 6); Scale bar, 50 μm. ( D ) At 120 days of age, MYH7 antibody immunostaining was performed on SOL, GAS, and PMM ( n = 3). ( E , F ) At 120 days of age, the mRNA expression levels of MYH7 and MYH4 in SOL, GAS, and PMM were detected using qRT-PCR ( n = 3). ( G ) Protein expression levels of MYH7 and MYH1 during the development of the SOL, GAS, and PMM were analyzed by Western Blot ( n = 3).

Article Snippet: Western blot analysis was performed with the following primary antibodies: MYH7 (1:1000, 22280-1-AP, Proteintech Group, Inc., Wuhan, China), MYH1 (1:10000, 67299-1-Ig, Proteintech Group, Inc., Wuhan, China), and Anti-GAPDH (1:10000, 10494-1-AP, Proteintech Group, Inc., Wuhan, China).

Techniques: Control, Staining, Immunostaining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: Expression levels of miR-199a-5p are decreased in human varicose vein tissues. (A) RT-qPCR revealed that the expression levels of VSMC differentiation biomarkers were decreased in varicose vein tissues (n=10). *P<0.05 vs. great saphenous vein tissues. (B) Western blot analysis revealed that the protein expression levels of VSMC differentiation biomarkers were decreased and the expression levels of FOXC2 were increased in varicose vein tissues (n=3). *P<0.05 vs. great saphenous vein tissues. (C) miR-199a-5p was downregulated and FOXC2 was upregulated in varicose vein tissues, as determined by RT-qPCR. (n=10). *P<0.05 vs. great saphenous vein tissues. miR-199a-5p, microRNA-199a-5p; FOXC2, forkhead box C2; MYH11, myosin heavy chain 11; RT-qPCR, Reverse transcription-quantitative PCR; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: miR-199a-5p regulates the expression levels of VSMC biomarkers. (A) Reverse transcription-quantitative PCR confirmed that miR-199a-5p was overexpressed and knocked down post-transfection with the miR-199a-5p mimics and inhibitor, respectively (n=3). (B) Overexpression or knockdown of miR-199a-5p promoted or inhibited the expression of VSMC differentiation biomarkers, respectively. *P<0.05 vs. control (n=10). (C) Western blot analysis of VSMC differentiation biomarkers. *P<0.05 vs. control (n=3). miR-199a-5p, microRNA-199a-5p; MYH11, myosin heavy chain 11; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Knockdown, Control, Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: FOXC2 rescue experiments. (A) RT-qPCR confirmed that FOXC2 was overexpressed and knocked down post-transfection with pcDNA3.1-FOXC2 vector or FOXC2 siRNA, respectively. *P<0.05 vs. control (n=3). (B) CCK-8 confirmed that VSMC proliferation was enhanced after transfection with miR-199a-5p mimics + FOXC2 vector compared with miR-199a-5p mimics alone. *P<0.05 (n=10). (C) Transwell migration assays revealed that FOXC2 enhanced VSMC migration. Magnification, ×40. *P<0.05 (n=10). (D) Western blot analysis revealed that the expression levels of VSMC differentiation biomarkers were decreased in cells transfected with miR-199a-5p mimics + FOXC2 vector compared with those in cells transfected with miR-199a-5p mimics only. *P<0.05 (n=3). (E) RT-qPCR revealed that FOXC3 decreased the expression levels of VSMC differentiation biomarkers compared with those in cells transfected with miR-199a-5p mimics only. *P<0.05 (n=10). (F) RT-qPCR was used to detect the expression levels of phenotypic transition biomarkers. *P<0.05 vs. control (n=10). (G) CCK-8 confirmed that proliferation of VSMCs was reduced in response to FOXC2 silencing, but increased in response to FOXC2 overexpression. *P<0.05 vs. control (n=3). (H) Wound healing assay revealed that migration of VSMCs was reduced post-transfection with the FOXC2 siRNA, but increased following the overexpression of FOXC2 compared with the control group. *P<0.05 vs. control (n=3). FOXC2, forkhead box C2; miR-199a-5p, microRNA-199a-5p; MYH11, myosin heavy chain 11; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; CCK-8, Cell Counting Kit-8.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Migration, Western Blot, Expressing, Over Expression, Wound Healing Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Cell Counting