5 bromo 2 deoxyuridine 5 brdu

Cardiomyocytes were isolated from 1- to 3-day old C57BL/6 mice according to the procedures described in our previous studies [23 (link), 53 (link)]. In brief, after the heart tissue was dissected and washed, it was finely minced and the chunks were placed in 0.25% trypsin. The pooled cell suspension was centrifuged and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The resuspension was cultured in a tissue culture flask for 90 min at 37°C to allow the fibroblasts to attach to the flask bottom [54 (link)]. The non-adherent and weakly attached cells, mainly cardiomyocytes, were removed and seeded into culture plates. Next, 5-bromo-2’-deoxyuridine (5-BrdU, 10 nM; #B5002; Sigma, Saint Louis, USA) was added to the culture medium to remove fibroblasts. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. After 48 h, cardiomyocytes that adhered to the culture dish were used for subsequent experiments [55 (link)]. Cells were deprived of serum and placed in an anoxic chamber for 12 h in a humidified atmosphere comprising 5% CO₂ and 95% N₂.

We used the following primary antibodies: mouse-anti-Rac1 (BD Transd. Laboratories, 610650), mouse-anti-p190A (BD Transd. Laboratories, 610149), mouse-anti-RhoA (26C4, Santa Cruz, sc-418), mouse–anti-RGS3 (CC-Q7, Santa Cruz, sc-100762), rabbit–anti-Tiam1 (C-16, Santa Cruz, sc-872), rabbit–anti-Gβ (T-20, Santa Cruz, sc-378), rabbit–anti-RGS3 (Abcam, ab2564), mouse–anti-c-myc (Klon 9E10, Oncogene), mouse–anti-β-actin (Sigma-Aldrich, A2228), mouse–anti-RhoA-GTP (Biomol). The corresponding horseradish peroxidase-conjugated secondary antibodies were from Sigma-Aldrich (Saint Louis, MO, USA, A-9044, A-9169), the fluorescent labelled secondary antibodies (anti-mouse-Alexa Fluor® 568-antibody, anti-rabbit-Alexa Fluor^® 633-antibody) were from Life Technologies. In this study, the following reagents and inhibitors were used: FGF-2 (Promega), carbamoylcholine chloride (carbachol), 5-bromo-2′-deoxyuridine (5-BrdU) (Sigma-Aldrich), PI3K-inhibitor LY294002 (2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; Alexis), pertussis toxin (PTX; Calbiochem), H1152P ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine, 2HCl, Merck).

The suppliers and the catalog numbers of the reagents are as follows. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, United States ; 22,400–089), fetal bovine serum (Life Technologies; 10,099), antibiotic-antimycotic (Life Technologies; 15,240–112), phosphate-buffered saline (PBS) (Life Technologies; 10010-049, pH7.4), trypsin-EDTA (Life Technologies; 25300-054, 0.05%), bovine serum albumin (Life Technologies; 15560012); D-Hanks solution (Beyotime, Jiangsu, China; C0218); type II collagenase (Sigma, St. Louis, MO, United States ; C6885); 5-bromo-2'-deoxyuridine (5-BrdU) (Sigma; B5002); Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan; CK04), IRDye 680CW (Licor, Lincoln, NE, United States ), protein molecular weight markers (Beyotime; P0066), BCA protein concentration determination kit (Solarbio, Beijing, China; PC0020). Antibodies: anti-glycogen synthase kinase beta (GSK3β) (Cell Signaling Technology (CST), Danvers, MA, United States ; 12,456), anti-AKT serine/threonine kinase 1 (AKT1) (CST; 2,967), anti-AKT serine/threonine kinase 2 (AKT2) (CST; 2,962), anti-β-catenin (Abcam, Cambridge, MA, United States ; ab24925), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abbkine, Wuhan, China; Abp57259).