Journal: Circulation Research

Article Title: Deletion of Yes-Associated Protein (YAP) Specifically in Cardiac and Vascular Smooth Muscle Cells Reveals a Crucial Role for YAP in Mouse Cardiovascular Development

doi: 10.1161/circresaha.114.303411

Figure Lengend Snippet: Figure 5. Deletion of yes-associated protein (YAP) induces a subset of cell cycle arrest gene expression without affecting expression of smooth muscle (SM)–specific genes. A, Total RNA was harvested from E15.5 control (open bars) YAP knockout (KO; hatched bars) dorsal aorta, and quantitative reverse transcription polymerase chain reaction was performed to assess gene expression as indicated. The level of gene expression in control vessels was set to 1 (solid line; n=5 to 7 embryos per genotype; *P<0.05). B, A representative Western blot using protein lysates from E15.5 control or YAP KO dorsal aorta tissue is shown. C, Immunoblot signals were normalized to α-tubulin loading control then expressed relative to signals from control aortae (set to 1, solid line; n=4 embryos per genotype; *P<0.05). D, Empty retrovirus or retrovirus encoding YAP was transduced into rat primary aortic SM cells (SMCs), and then G-protein–coupled receptor 132 (Gpr132) expression was analyzed by Western blotting. An arrow points to the endogenous YAP band. Ccnd1 indicates cyclin d1; Hic-5, hydrogen peroxide–inducible clone 5; MLCK, myosin light chain kinase; PCNA, proliferating cell nuclear antigen; Sfn stratifin; and Trp63, transformation related protein 63.

Article Snippet: Antibodies used in this study were: YAP (Sigma, mouse, 1:2000), SM α- actin (Sigma, AC-74, 1:4000), calponin (Santa Cruz, N-15R, 1:1000), Hic-5 (BD, 1:5000), MLCK (Sigma, clone K36, 1:5000), Gpr132 (Proteintech, rabbit, 1:2000), Trp63 (Santa Cruz, 4A4, mouse, 1:1000), stratifin (Sfn, Millipore, AB9742, rabbit, 1:2000), PCNA (Santa Cruz, 1:500), cyclin D1 (Ccnd1, Cell signaling, #2926, 1:1000), α-tubulin (Cell Signaling, 1:5000), GAPDH (Santa Cruz, 1:2000), TEAD1 (BD, 1:2000), HDAC4 (Millipore, 1:2000).

Techniques: Gene Expression, Expressing, Control, Knock-Out, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Transformation Assay

Journal: Circulation Research

Article Title: Deletion of Yes-Associated Protein (YAP) Specifically in Cardiac and Vascular Smooth Muscle Cells Reveals a Crucial Role for YAP in Mouse Cardiovascular Development

doi: 10.1161/circresaha.114.303411

Figure Lengend Snippet: Figure 6. G-protein–coupled receptor 132 (Gpr132) suppresses smooth muscle cell (SMC) growth. A, After transfected with control or Gpr132 small interfering RNA duplexes, rat primary aortic SMCs were incubated with bromodeoxyuridine (BrdU) for 24 hours, and then immunostaining was performed to BrdU-positive SMCs by using an anti-BrdU antibody (red) and costained with 4',6-diamidino-2-phenylindole(blue) for nuclei (scale bar, 50 μm). B, The fraction of BrdU-positive SMCs was counted and plotted. C, After silencing Gpr132, rat primary aortic SMCs were seeded at equal density at day (D) 0 and counted at each time point as indicated (n=3; *P<0.05). D, After transduction with Gpr132 or control green fluorescent protein (GFP) adenovirus, rat primary aortic SMCs were harvested for propidium iodide staining to analyze cell cycle by a flow cytometry (*P<0.05). E, After infection with Gpr132 or GFP control adenovirus, rat primary aortic SMCs were seeded at equal density and counted at each time point indicated (*P<0.05). F, E15.5 dorsal aortic SMCs from yes-associated protein knockout (KO) or control embryos were transfected with scrambled control or Gpr132 silencing duplexes. After 24 hours, cells then were plated and counted at each time point as indicated (*P<0.05, control-siControl vs control- siGpr132; #P<0.05, KO-siControl vs KO-siGpr132).

Article Snippet: Antibodies used in this study were: YAP (Sigma, mouse, 1:2000), SM α- actin (Sigma, AC-74, 1:4000), calponin (Santa Cruz, N-15R, 1:1000), Hic-5 (BD, 1:5000), MLCK (Sigma, clone K36, 1:5000), Gpr132 (Proteintech, rabbit, 1:2000), Trp63 (Santa Cruz, 4A4, mouse, 1:1000), stratifin (Sfn, Millipore, AB9742, rabbit, 1:2000), PCNA (Santa Cruz, 1:500), cyclin D1 (Ccnd1, Cell signaling, #2926, 1:1000), α-tubulin (Cell Signaling, 1:5000), GAPDH (Santa Cruz, 1:2000), TEAD1 (BD, 1:2000), HDAC4 (Millipore, 1:2000).

Techniques: Transfection, Control, Small Interfering RNA, Incubation, Immunostaining, Transduction, Staining, Flow Cytometry, Infection, Knock-Out

Journal: Circulation Research

Article Title: Deletion of Yes-Associated Protein (YAP) Specifically in Cardiac and Vascular Smooth Muscle Cells Reveals a Crucial Role for YAP in Mouse Cardiovascular Development

doi: 10.1161/circresaha.114.303411

Figure Lengend Snippet: Figure 7. Yes-associated protein (YAP) recruits histone deacetylase-4 (HDAC4) to a muscle CAT element (MCAT) element to suppress G-protein–coupled receptor 132 (Gpr132) promoter activity. A, Schematic diagram of the Gpr132 gene structure showing a conserved MCAT element within intron 1. A, Wild-type or MCAT mutant reporter (mutated nucleotide in MCAT element is underscored) containing a 1.5k bp fragment within Gpr132 gene intron 1 was generated into pGL2E luciferase vector. The location of 2 sets of primers (PS), localized either in intron 1 or exon (E) 3 used for chromatin immunoprecipitation (ChIP) assays in C, is shown. B, Wild-type or MCAT mutant Gpr132 promoter reporter genes were transfected into pulmonary arterial cultured-1 smooth muscle cells (SMCs) together with YAP in the absence or presence of HDAC4 expression plasmid. Values are presented as relative luciferase activity compared with control vector (set to 1) and are the mean±SE of 6 samples. C, Adenovirus expressing green fluorescent protein (GFP) or Flag-tagged YAP was transduced into rat primary rat aortic SMCs, and then chromatin was harvested for immunoprecipitation with anti-Flag antibody. The precipitated DNA was amplified by a real-time polymerase chain reaction (PCR) using Gpr132 gene-specific primers that span the intronic MCAT region (primer set [PS]1) or exon 3 (PS2) as depicted in A. The YAP binding is expressed relative to GFP control sample (set to 1; *P<0.05). D, ChIP assay was performed as described in C, except that anti-HDAC4 antibody was used to immunoprecipitate endogenous HDAC4 and real-time PCR was performed using PS1 primers (*P<0.05). E, Adenovirus expressing YAP was transduced into rat aortic SMCs, and endogenous HDAC4 was immunoprecipitated by using anti-HDAC4 antibody. Western blot was then performed by using the indicated antibodies. A species- matched IgG served as control. F, Chromatin was harvested from SMCs overexpressing YAP. The chromatin was immunoprecipitated first with anti-YAP antibody, eluted, and then immunoprecipitated with either IgG or anti-HDAC4 antibody. Immunoprecipitated DNA was then analyzed by quantitative PCR using PS1 primers (*P<0.05). G, Schematic diagram depicting the findings of this study.

Article Snippet: Antibodies used in this study were: YAP (Sigma, mouse, 1:2000), SM α- actin (Sigma, AC-74, 1:4000), calponin (Santa Cruz, N-15R, 1:1000), Hic-5 (BD, 1:5000), MLCK (Sigma, clone K36, 1:5000), Gpr132 (Proteintech, rabbit, 1:2000), Trp63 (Santa Cruz, 4A4, mouse, 1:1000), stratifin (Sfn, Millipore, AB9742, rabbit, 1:2000), PCNA (Santa Cruz, 1:500), cyclin D1 (Ccnd1, Cell signaling, #2926, 1:1000), α-tubulin (Cell Signaling, 1:5000), GAPDH (Santa Cruz, 1:2000), TEAD1 (BD, 1:2000), HDAC4 (Millipore, 1:2000).

Techniques: Histone Deacetylase Assay, Activity Assay, Mutagenesis, Generated, Luciferase, Plasmid Preparation, Chromatin Immunoprecipitation, Transfection, Cell Culture, Expressing, Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot