rat aortic smcs (rasmcs)  (Lonza)

Journal: Biomolecules

Article Title: Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

doi: 10.3390/biom14060709

Figure Lengend Snippet: Nucleolin localization and iSN04 incorporation in A10 cells. ( A ) Representative fluorescence images of nucleolin staining of A10 cells in GM (day 0) and DM with or without 10 μM iSN04 (day 4). Scale bar, 50 μm. ( B ) Representative fluorescence images of A10 cells treated with 5 μg/mL 6-FAM-iSN04 in GM. Scale bar, 50 μm. 6-FAM, 6-carboxyfluorescein; DAPI, 4′,6-diamidino-2-phenylindole; DM, differentiation medium; GM, growth medium.

Article Snippet: Embryonic rat thoracic aortic SMC line A10 (CRL-1476; ATCC, Manassas, VA, USA) was maintained in growth medium (GM) consisting of DMEM (Nacalai, Osaka, Japan), 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), and a mixture of 100 units/mL penicillin and 100 μg/mL streptomycin (P/S) (Nacalai).

Techniques: Fluorescence, Staining

Journal: Biomolecules

Article Title: Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

doi: 10.3390/biom14060709

Figure Lengend Snippet: The effect of iSN04 on proliferation and differentiation of A10 cells. ( A ) Representative fluorescence images of EdU staining of A10 cells pre-treated with 30 μM iSN04 in GM for 24 h and then with 10 μM EdU in GM for 3 h. Scale bar, 200 μm. The ratio of EdU + cells was quantified. * p < 0.05 vs. control (Student’s t -test). n = 4. ( B ) Representative fluorescence images of α-SMA staining of A10 cells treated with 3 or 10 μM iSN04 in DM for 4 days. Scale bar, 200 μm. α-SMA signal intensities per number of DAPI + nuclei were quantified. ** p < 0.01 vs. control; †† p < 0.01 vs. 3 μM iSN04 (Scheffe’s F test). n = 7. DAPI, 4′,6-diamidino-2-phenylindole; DM, differentiation medium; EdU, 5-ethynyl-2′-deoxyuridine; GM, growth medium; α-SMA, α-smooth muscle actin.

Article Snippet: Embryonic rat thoracic aortic SMC line A10 (CRL-1476; ATCC, Manassas, VA, USA) was maintained in growth medium (GM) consisting of DMEM (Nacalai, Osaka, Japan), 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), and a mixture of 100 units/mL penicillin and 100 μg/mL streptomycin (P/S) (Nacalai).

Techniques: Fluorescence, Staining, Control

Journal: The Journal of Clinical Investigation

Article Title: BAF60c prevents abdominal aortic aneurysm formation through epigenetic control of vascular smooth muscle cell homeostasis

doi: 10.1172/JCI158309

Figure Lengend Snippet: ( A ) Luciferase assays in A7r5 cells transfected with MYH11 , ACTA2 , or TAGLN promoter-driven luciferase reporters and infected with Ad- GFP or Ad- BAF60c (10 MOI). After 48 hours, the promoter activities were determined by dual luciferase assay and normalized by Renilla activity ( n = 4). ( B ) A7r5 cells were transfected with siControl or si Baf60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for 24 hours. ChIP assays were performed to determine SRF-binding activity to Myh11 , Acta2 , Cnn1 , and Tagln promoters. IgG served as control. ( C and D ) HASMCs were transfected with siControl or si BAF60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for 24 hours and ChIP-Seq was performed using BRG1, H3K9ac, and H3K27ac Abs. ( C ) Histogram of ChIP-Seq reads of H3K9ac ±3 Kb surrounding the TSS of genes (left). Quantification of relative read ratio of the H3K9ac signals within the TSS ± 3 Kb is presented in box plots (right). ( D ) IGV image showing localization of BRG1 and H3K9ac and H3K27ac within MYH11 promoter in HASMCs. ( E ) BRG1 and H3K9ac binding on the predicted SRF-binding site located in the human MYH11 promoter were determined by ChIP assay in HASMCs transfected with siControl or si BAF60c (30 nM) for 48 hours, followed by serum starvation in Opti-MEM for 24 hours. ( F – H ) HASMCs were transfected with siControl or si BAF60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for another 24 hours, and nuclear proteins were isolated and subjected to Co-IP using BRG1, SRF, or P300 Abs. IgG was the negative control. Three independent experiments were performed for B and E – H . Data are represented as mean ± SEM. Two-way ANOVA followed by Holm-Šidák post hoc analysis ( A , B , and D ); Mann-Whitney U test ( C ); paired t test ( F – H ).

Article Snippet: The rat embryonic thoracic aortic SMC line A7r5 was purchased from ATCC and cultured in DMEM/Nutrient Mixture F-12 (DMEM/F12) (Gibco, Thermo Fisher Scientific; 11320-033) containing 10% FBS (Thermo Fisher Scientific) and 50 mg/mL of a penicillin/streptomycin solution.

Techniques: Luciferase, Transfection, Infection, Activity Assay, Binding Assay, Control, ChIP-sequencing, Isolation, Co-Immunoprecipitation Assay, Negative Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: BAF60c prevents abdominal aortic aneurysm formation through epigenetic control of vascular smooth muscle cell homeostasis

doi: 10.1172/JCI158309

Figure Lengend Snippet: ( A ) Representative images of TUNEL staining (green) and quantification of apoptotic HASMCs transfected with siControl or si BAF60c (30 nM) for 48 hours and subsequently stimulated for 24 hours with 200 μM hydrogen peroxide (H 2 O 2 ). Scale bars: 20 μm. ( B – E ) HASMCs were transfected with siControl and si BAF60c (30 nM) ( B and C ) or infected with Ad- GFP and Ad- BAF60c (10MOI) ( D and E ). After 48 hours, cells were stimulated with 200 μM H 2 O 2 for 24 hours. Expression of BAF60c and BCL2 was assessed by qPCR ( B and D , n = 6) and Western blot ( C and E , n = 4). ( F ) HASMCs were transfected with siControl or si BAF60c . After 48 hours, cells were serum starved in Opti-MEM for 24 hours, and then ChIP-Seq was performed with Abs against BRG1, H3K9ac, and H3K27ac. IGV image showing BRG1, H3K9ac, and H3K27ac ChIP-Seq coverage at peaks within human BCL2 gene promoter. ( G ) BRG1 and KLF5 binding to the predicted KLF-binding sites located in the BCL2 promoter was determined by ChIP assay in HASMCs transfected with siControl or si BAF60c (30 nM) for 48 hours and serum starved in Opti-MEM for 24 hours. ( H ) A7r5 cells were transfected with WT or Del (region deleted) luciferase reporter driven by BCL2 promoter and then infected with Ad- lacZ or Ad- BAF60c . After 48 hours, promoter activities were determined, and results are presented relative to A7r5 transfected with WT and infected with Ad- lacZ group ( n = 8/group). ( I ) HASMCs were transfected with siControl or si BAF60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM media for 24 hours, followed by nuclear protein isolation and Co-IP using BRG1 or KLF5 Abs. IgG was negative control. Three independent experiments were performed for G and I . Data are represented as mean ± SEM. Two-way ANOVA followed by Holm-Šidák post hoc analysis ( A – E and G – H ); paired t test ( I ).

Article Snippet: The rat embryonic thoracic aortic SMC line A7r5 was purchased from ATCC and cultured in DMEM/Nutrient Mixture F-12 (DMEM/F12) (Gibco, Thermo Fisher Scientific; 11320-033) containing 10% FBS (Thermo Fisher Scientific) and 50 mg/mL of a penicillin/streptomycin solution.

Techniques: TUNEL Assay, Staining, Transfection, Infection, Expressing, Western Blot, ChIP-sequencing, Binding Assay, Luciferase, Isolation, Co-Immunoprecipitation Assay, Negative Control