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: 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: For MYH11 KD, control construct (sc-418922) and MYH11 CRISPR/Casp KO plasmids (sc-400695) were purchased from Santa Cruz Biotechnology.

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: For MYH11 KD, control construct (sc-418922) and MYH11 CRISPR/Casp KO plasmids (sc-400695) were purchased from Santa Cruz Biotechnology.

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: For MYH11 KD, control construct (sc-418922) and MYH11 CRISPR/Casp KO plasmids (sc-400695) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Fluorescence

Journal: Cell Stem Cell

Article Title: The Dynamic Transcriptional Cell Atlas of Testis Development during Human Puberty

doi: 10.1016/j.stem.2019.12.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MYH11 Dilution: 1:100 , Atlas Antibodies , cat# HPA015310, RRID: AB_1854261.

Techniques: Immunohistochemistry, Software

Journal: Scientific Reports

Article Title: RhoA accelerates atherosclerosis progression by interacting with Hspa5

doi: 10.1038/s41598-025-33741-w

Figure Lengend Snippet: RhoA is highly expressed in the vascular smooth muscle of mice with AS. ( A ) DEGs were predicted using the GSE215969 microarray and shown using a volcano map. Red: upregulated genes; blue: downregulated genes; gray: no significant difference. ( B ) DEGs from the GSE215969 microarray were also shown using a heat map. Red: upregulation; blue: downregulation. AS mouse model was established using high-fat diet, while the control group mice were fed standard chow. ( C) Representive images of en face Oil Red O staining assay. Scale bar = 2 mm. The serum was collected from mice, and ELISA was conducted to measure the levels of ( D ) IL-1β and ( E ) IL-6. After collecting the aortic root from mice, ( F ) the location and expression of RhoA were visualized using IF, and SM-MHC was stained to show the smooth muscle cells. Scale bar = 100 μm. ( G ) RhoA activity was detected using western blotting ater Rhotekin pull-down assay, and the results were quantified in (H). n = 6/group.

Article Snippet: Following blocking with 5% bovine serum albumin (BSA; MCE, Monmouth Junction, NJ, USA), sections were incubated with primary antibodies against SM-MHC (60222-1-Ig and 21404-1-AP, Proteintech, Wuhan, China), RhoA (ab187027, Abcam, Cambridge, MA, USA), and collagen (ab34710, Abcam) overnight at 4 °C.

Techniques: Microarray, Control, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Western Blot, Pull Down Assay

Journal: Scientific Reports

Article Title: RhoA accelerates atherosclerosis progression by interacting with Hspa5

doi: 10.1038/s41598-025-33741-w

Figure Lengend Snippet: Silencing of RhoA attenuates plaque lesions and inflammatory response in AS mice. AS mouse model was established using high-fat diet, and AAV-shRhoA, AAV-shNC, AAV-NC, and AAV-shHspa5 were injected through the tail vein. ( A ) Hspa5 expression was measured using qPCR. The whole aorta was obtained from mice, and ( B ) representive images of en face Oil Red O staining. Scale bar = 2 mm. The aortic root was obtained from mice, and ( C ) Oil Red O staining was performed to determine plaque lesions, and ( D ) the staining area was quantified. Scale bar = 200 μm. ( E ) Representive images of IF staining to observe smooth muscle cells (SM-MHC staining) and collagen (collagen staining). Scale bar = 50 μm. ( F ) MMP9 and ( G ) Lp-PLA2 expression was detected using qPCR. The serum was collected from mice, and the levels of ( H ) IL-1β, ( I ) IL-6, and ( J ) TNF-α were measured by ELISA. n = 6/group.

Article Snippet: Following blocking with 5% bovine serum albumin (BSA; MCE, Monmouth Junction, NJ, USA), sections were incubated with primary antibodies against SM-MHC (60222-1-Ig and 21404-1-AP, Proteintech, Wuhan, China), RhoA (ab187027, Abcam, Cambridge, MA, USA), and collagen (ab34710, Abcam) overnight at 4 °C.

Techniques: Injection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A : Representative fluorescence microscopy images and quantification of MitoSOX fluorescence in VSMCs treated with either a vehicle or 100 µM Ang II for 30 min. The scale bar is 2 µm. Data represent the integrated density of MitoSOX fluorescence (mean ± SEM, n = 8). B : The amount of 8-OHdG was measured by ELISA in total DNA from VSMCs treated with a vehicle or 100 µM Ang II for 24 h (mean ± SEM, n = 4). C : The oxygen consumption rate (OCR) was measured in VSMCs treated with a vehicle or Ang II for 24 h using an Agilent Seahorse XF96 analyzer (mean ± SEM, n = 12). D : Mitochondrial maximal respiration capacity was derived from OCR measurements in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n = 10). E : Mitochondrial spare capacity was also derived from OCR measurements in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n= 10). F : Flow cytometry analysis and quantification of MYH11 + TAGLN + cells in VSMCs treated with either a vehicle or Ang II for 24 h (mean±SEM, n=10). G : Flow cytometry analysis and quantification of CD11b + CD68 + cells in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n = 10).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A : Western blot analysis of CGAS and STING expression in protein lysates from vehicle or Ang II-treated VSMCs isolated from wild-type, Nox4 TG, and Nox4 -/- mice. B : The quantification of CGAS protein expression in vehicle or Ang II-treated VSMCs. The data is fluorescence intensity fold change over vehicle- treated wild-type cells, adjusted for TUBB levels (mean ± SEM, n = 4). C : CGAMP levels were measured using ELISA in cell lysates from vehicle or Ang II-treated VSMCs (mean ± SEM, n = 4). D : The quantification of STING protein expression was analyzed with Western blot in vehicle or Ang II-treated VSMCs. Data are presented as fluorescence intensity fold change over vehicle-treated wild-type cells, adjusted for TUBB levels (mean ± SEM, n=4). E : Western blot analysis and quantification of CGAS protein levels in the abdominal aorta protein lysates from Apoe -/- , Nox4 TG/ Apoe -/- , and Nox4 -/- / Apoe -/- mice treated with Ang II. STING protein expression was quantified with Western blot in vehicle or Ang II-treated VSMCs. Data are presented as fluorescence intensity fold change over Apoe -/- mice, adjusted for ACTB levels (mean ± SEM, n = 4). F : Western blot analysis and quantification of STING and phospho-STING levels were performed on abdominal aorta protein lysates from Apoe -/- , Nox4 TG/ Apoe -/- , and Nox4 -/- / Apoe -/- mice treated with Ang II. Data are presented as fluorescence intensity fold change over Apoe -/- mice, adjusted for ACTB levels (mean ± SEM, n = 4). G & H : Representative fluorescence microscopy images and quantification of CGAS ( G ) and STING ( H ) expression in human control aorta and AAA frozen sections. These sections were stained for immunoreactive CGAS ( G ) or STING ( H ) (red) and MYH11 (green), with DAPI (blue) used for counterstaining. The scale bar is 100 µm. Data are presented as fluorescence integrated density (mean ± SEM, n = 9).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Western Blot, Expressing, Isolation, Fluorescence, Enzyme-linked Immunosorbent Assay, Microscopy, Control, Staining

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A & B : Flow cytometry analysis of single-cell suspension from the abdominal aorta of mice treated with Ang II. Panel A presents the quantification of MYH11 + cells, while Panel B shows the quantification of CD45 + CD68 + CD11b + cells. The data represent the cell fraction of all aortic cells (mean ± SEM, n=6). C : Flow cytometry analysis and quantification of abdominal aorta single-cell suspension from Ang II-treated mice showing the proportion of MYH11 + cells expressing macrophage markers. This data shows the fraction of CD68 + CD11b + cells as a percentage of MYH11 + cells (mean ± SEM, n = 6). D : A t-SNE clustering analysis of flow cytometry data from the abdominal aorta of Ang II-treated mice was performed. MYH11 + cells were clustered based on the expression of CNN1, ACTA2, TAGLN, CD45, CD68, CD11b, CD38, TNFα, IL1b, and IL6, resulting in distinct clusters presented on the t-SNE plot. E : A heat map representation illustrates the relative expression of SMC, macrophage, and inflammatory markers based on the mean fluorescence intensity for each cluster. F & H : The relative abundance of distinct clusters as a proportion of MYH11 + cells (mean ± SEM, n = 6).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Flow Cytometry, Suspension, Expressing, 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: