Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: Smooth muscle myosin localizes at the tip of lamellipodia. ( A ) Human airway smooth muscle (HASM) cells were plated onto collagen-coated coverslips for 30 min and stained for 20-kDa myosin light chain (MLC 20 ) and F-actin. MLC 20 is found at the leading cell edge. In addition, F-actin is also localized at the cell edge. ( B ) Myosin-11 (MYH11) colocalizes with MLC 20 at the leading edge. ( C ) Phosphorylated MLC 20 (pMLC 20 ) is found to position at the leading edge. The arrows point to the leading edge. Scale bar: 10 µm.

Article Snippet: Myosin heavy chain (MYH11) antibody was purchased from Santa Cruz Biotechnology (SC-6956, L/N C1815).

Techniques: Staining

Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: Knockdown (KD) of MLC 20 or MYH11 attenuates cell migration. ( A ) Human airway smooth muscle (HASM) cells treated with control (Ctrl) siRNA or MLC 20 siRNA were evaluated by immunoblot analysis. MLC siRNA reduces the expression of MLC 20 in HASM cells. Data are mean values of experiments from five cultures from three donors. Error bars indicate SD. ( B ) The migration of HASM cells was examined by using the wound healing assay. n = six experiments from three donors. Error bars indicate SD. ( C ) Cells were treated with Ctrl or Crispr/Cas9 constructs followed by immunoblot analysis. Data are the mean values of experiments from four cultures from three donors. Error bars indicate SD. ( D ) MYH11 KD reduces the migration of HASM cells. n = six experiments from three donors. Error bars indicate SD. ** p < 0.01. Student’s t -test was used for statistical analysis.

Article Snippet: Myosin heavy chain (MYH11) antibody was purchased from Santa Cruz Biotechnology (SC-6956, L/N C1815).

Techniques: Knockdown, Migration, Control, Western Blot, Expressing, Wound Healing Assay, CRISPR, Construct

Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: MYH11 orchestrates the positioning of c-Ab, cortactin, Pfn-1, and Abi1 to the cell edge. ( A ) Ctrl and MYH11 KD cells were plated onto collagen-coated coverslips for 30 min followed by immunofluorescence and fluorescence analysis. The arrows point to the leading edge. Scale bar: 10 µm. ( B ) Data are mean values of experiments from at least 20 cells for each group. Error bars indicate SD. One-way ANOVA was used for statistical analysis. ** p < 0.01; * p < 0.05.

Article Snippet: Myosin heavy chain (MYH11) antibody was purchased from Santa Cruz Biotechnology (SC-6956, L/N C1815).

Techniques: Immunofluorescence, Fluorescence

Journal: PLoS ONE

Article Title: Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

doi: 10.1371/journal.pone.0184228

Figure Lengend Snippet: A . MYH11 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2, ++++ P < 0.05 versus MCa1. B . MUC1 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2, ++++ P < 0.05 versus MCa1. Normal Mammary Epithelium (NME), Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Article Snippet: The phenotype characterization of the mixed cell cultures was performed by qPCR using oligos for MUC1 (Cf02626760_m1, Cf02680908_s1) an epithelial marker, and MYH11 (Cf02697091_m1, Cf02632489_m1) a smooth muscle marker.

Techniques:

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

Journal: Cells

Article Title: Biomechanical Assessment of Macro-Calcification in Human Carotid Atherosclerosis and Its Impact on Smooth Muscle Cell Phenotype

doi: 10.3390/cells11203279

Figure Lengend Snippet: High stretch induces SMC alignment but downregulates most markers of differentiated SMCs. ( A ) Representative images of vascular SMCs after 8 h on stretched/unstretched silicone chambers or rigid culture plates. ( B ) ACTA2/smooth muscle actin as well as LMOD1 were upregulated in cells grown on tissue culture plates as compared to silicon membranes. However, MYH11 showed the opposite trend. Representative images show 24 h. ( C ) MYOCD , TAGLN and CNN1 transcript levels were elevated after 24 h in SMCs grown on rigid plates, while PDLIM7 mRNA expression was most highly upregulated after 24 h under non-stretch conditions. Images show ( A ) 4×/20× magnification and ( B ) 63×. Insets show corresponding isotype negative control. Plots show mean with SEM. Statistical difference assessed by two-way ANOVA. Expression levels calculated according to ΔΔCT and normalized to unstretched conditions at 8 h.

Article Snippet: PCR amplification was performed in 384-well plates in a 7900 HT real-time PCR system (Applied Biosystems, Carlsbad, CA, USA), using TaqMan ® Universal PCR Master Mix (#4324018, Applied Biosystems, Carlsbad, CA, USA) and TaqMan ® Gene Expression Assays (MYOCD probe Hs00538076_m1; ACTA2 probe Hs00426835_g1; MYH11 probe Hs00975796_m1; CNN1 probe Hs00959434_m1; LMOD1 probe Hs00201704_m1; PDLIM7 probe Hs00193775_m1; HSPG2 probe Hs01078536 m1; TAGLN probe Hs01038773_g1; Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Negative Control

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: MYH11 , MYOSIN, HEAVY CHAIN 11, SMOOTH MUSCLE , 16p13.11 , 15704492 , 153896 , Hs00358138_cn , NS , 160745 , 16444274.

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Case reports of likely causal CNVs

Article Snippet: MYH11 , MYOSIN, HEAVY CHAIN 11, SMOOTH MUSCLE , 16p13.11 , 15704492 , 153896 , Hs00358138_cn , NS , 160745 , 16444274.

Techniques: