Gata4

Whole cell lysates were harvested by using lysis buffer (20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and 1 mg/mL leupeptin) on a rotation wheel for 1 h at 4°C. After centrifugation at 10,000 × g for 10min, the supernatant was prepared as a protein extract. Equal concentrations of proteins were fractionated by electrophoresis on 8%-12% acrylamide gels and were transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany), followed by blotting with antibodies for GATA4 (Abcam, Cambridge, MA, USA), YAP (Cell Signaling Technology, MA, USA), p21 (Santa Cruz Biotech, Dallas, TX, USA), GSK3β (Santa Cruz Biotech, Dallas, TX, USA), Phospho-GSK3β (Cell Signaling Technology, MA, USA), Akt (Cell Signaling Technology, MA, USA), phospho-Akt (Cell Signaling Technology, MA, USA), β-catenin (Santa Cruz Biotech, Dallas, TX, USA), Krüppel-like factor 4 (KLF4, Abcam, Cambridge, MA, USA), glyceraldehyde 3-phosphoate dehydrogenase (GAPDH, Santa Cruz Biotech, Dallas, TX, USA), and β-actin (Sigma-Aldrich, USA) followed by secondary staining with horseradish peroxidase-conjugated immunoglobulin G. Protein expression was detected by using an Image Reader (LAS-3000 Imaging System, Fuji Photo Film, Tokyo, Japan). The expression level was quantified with Image J (NIH, Bethesda, MD, USA).

Proteins were extracted and quantified as previously reported [18 (link)]. Each sample containing equal amounts of protein (30 μg) was subjected to SDS-PAGE and then was electro-blotted onto PVDF membranes. Immunoblotting and detection of the GATA4(1:1000, Abcam, English), Nkx2.5(1:1000,Abcam, English) and cTnI(1:5000) expression were performed. The immunoreactive bands were revealed with enhanced chemiluminescence (Millipore, Billerica, USA), and analyzed using Quantity One Version 4.62 software (Bio-Rad, Richmond, CA). The β-actin was used as the internal controls.

Cells were fixed with freshly prepared 4% PFA and probed with the following antibodies – NG2 (1:100, Millipore), PDGFRβ (1:50, Santa Cruz), GATA4 (1:100), (all Abcam) followed by Alexafluor 488 anti-rabbit or anti-mouse secondary antibodies (Invitrogen). Always, isotype negative controls were performed to ensure the immunofluorescence specificity. Cell nuclei were stained with 300 nM 4′,6-diamidino-2-phenylindole dilactate (DAPI) (ThermoFisher Scientific). Images were acquired with a fluorescent microscope (Olympus BX40) at 40x magnification and merged using ImageJ software.

The procedure and protocol have been described in our previous reports [34 (link),35 (link),36 (link)]. Re-hydrated paraffin sections were treated with 3% H₂O₂ for 30 min and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies against CD31 (1:100, Bio-Rad, Hercules, CA, USA), vascular endothelial growth factor (VEGF) (1:400, Abcam Inc., Cambridge, MA, USA), C-kit (1:100, Santa Cruz), GATA4 (1:500, Abcam) and SOX2 (1:100, Abcam). Three sections of heart specimens from each rat were analyzed. For quantification, three randomly selected HPFs (400× for IHC and IF studies) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined by summation of all numbers divided by 9.

ChIP experiments were conducted using a ChIP assay kit (ChIP Kit-one step, Abcam, USA). After the homogenization of cardiac tissues, 1% formaldehyde (SigmaAldrich, St. Louis, MO, USA) was added to the samples to cross-link proteins to DNA. The cross-linked DNA was then fragmented into small fragments (500–1000 bp) using an ultrasound (Bioruptor UCD-200). Next, the protein–DNA complexes were precipitated using monoclonal antibodies specific for acetylated histone 3 (AcH3), lysine 4 in H3 (AcH3K4), lysine 9 in H3 (AcH3K9), HDAC1, GATA4, and Mef2c (Abcam, Cambridge, USA). Anti-RNA polymerase antibodies were used as a positive control and anti-mouse IgG was used as a negative control. The total column input also served as a positive control. Next, cross-linked protein–DNA complexes were removed and the DNA was extracted. Specific quantitative real-time PCR (qPCR) primers were designed to determine levels of AcH3, AcH3K4, and AcH3K9 near the proximal promoter regions of Atp2a2. The qPCR primer sequences used to amplify the SERCA2a promoter were as follows: 5′-AGCCAAGGACACCAGTGC-3′ and 5′-GGGATAGAGCGCGGAGTT- 3′.