Significance of expression differences between and samples was determined using Student’s test. epigenetic silencing of specifically within the second heart field-derived mesenchymal cells and thereby promotes termination of EndMT. Genetic deletion of in the murine ROCK inhibitor second heart field results in increased TGF- bioavailability within mesenchymal cells, perpetual activation of mesenchymal cells, aberrant EndMT, ROCK inhibitor and altered extracellular matrix homeostasis, observed in patients with semilunar valve pathologies. Together, these results uncover that epigenetic silencing mediated by HDAC3 in a deacetylase-independent manner orchestrates second heart field development, which may be a molecular target in human cardiovascular anomalies. Experimental Procedures Mice Transgenic mice were obtained from the Jackson Laboratories. The University of Massachusetts Medical School Institutional Animal Care and Use Committee approved all animal protocols. Histology Tissue samples were fixed in 2% paraformaldehyde at 4 C overnight, ethanol-dehydrated, embedded in paraffin, and sectioned at 6C8-m thickness using a microtome. Antibodies and Reagents The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- pan-specific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor? 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories). Recombinant TGF- was purchased from R&D Systems. Alcian blue, alkaline alcohol, orcein, alcoholic hematoxylin, ferric chloride, Lugol’s iodine, woodstain scarlet acid fuchsin, phosphotungstic acid, saffron, Bouin’s fixative, Weigert’s iron hematoxylin A, Weigert’s iron hematoxylin B, phosphomolybdic acid-phosphotungstic acid, aniline blue, and Van Gieson’s solution were purchased from Electron Microscopy Sciences. Harris modified hematoxylin, eosin Y, ethanol, xylenes, glacial acetic acid, paraformaldehyde, paraffin, potassium ferricyanide, potassium ferrocyanide, and deoxycholic acid were purchased from Fisher. Polyethylenimine, linear, was purchased from Polysciences. X-gal was purchased from 5 Prime. Vectashield mounting medium, ROCK inhibitor the Vectastain Elite ABC kit, and the DAB Peroxidase Substrate kit were purchased from Vector Laboratories. The RNeasy minikit and GST bead slurry were purchased from Qiagen. Power SYBR Green PCR Master Mix, Superscript first strand synthesis kit, TOPO-TA cloning kit, DMEM high glucose with sodium pyruvate, penicillin/streptomycin, and horse serum were purchased from Invitrogen. The CellsDirectTM one-step quantitative RT-PCR kit, insulin-transferrin-selenium, Epoxy M-450 Dynabeads, ROCK inhibitor and TRIzol were purchased from Life Technologies, Inc. Rat tail collagen type I was purchased from BD Biosciences. iScript reverse transcription supermix was purchased from Bio-Rad. The sandwich ELISA assay kit for TGF-1 was purchased from R&D Systems. The sandwich ELISA assay kit for phospho-SMAD2/3 was purchased from Cell Signaling. The QuikChange II XL site-directed mutagenesis kit was purchased from Stratagene. Passive lysis buffer and the Dual-Luciferase reporter assay kit were purchased from Promega. Fetal bovine serum, donkey serum, gelatin, and magnetic anti-FLAG beads were purchased from Sigma. Agarose-IgG and IgA bead slurry were purchased from Santa Cruz Biotechnology and Life Technologies. The EZ-ChIP assay kit and HDAC assay kit were purchased from Millipore. The TaKaRa DNA ligation kit was purchased from Clontech. Hematoxylin and Eosin Staining Hematoxylin and eosin staining was performed by deparaffinizing sections in xylenes, rehydrating through an ethanol gradient, 30-s or 2-min stain with 30% or 100% Harris modified hematoxylin, and a 30-s counterstain with eosin Y. Slides were rinsed and dehydrated with ethanol, cleared with xylenes, and mounted with Vectashield mounting CREB4 medium. Movat’s Pentachrome Staining Movat’s pentachrome staining was conducted by deparaffinizing and rehydrating slides, followed by a 20-min stain in Alcian blue, a 1-h differentiation in alkaline alcohol, a 20-min stain in Orcein-Verhoeff solution (Orcein, alcoholic hematoxylin, ferric chloride, and Lugol’s iodine), a 2-min stain with woodstain scarlet acid fuchsin, a rinse in acetic acid, and a 10-min differentiation in 5% phosphotungstic acid, followed by a 15-min stain in saffron. Sections were dehydrated in ethanol, cleared in xylenes, and mounted with Vectashield mounting medium. Masson’s Trichrome Staining Masson’s trichrome staining was performed by deparaffinizing and rehydrating sections through an ethanol gradient followed by a.
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