Connexin 43

Shikonin, Aconitine and Notoginsenoside R1, 98% or higher purity (HPLC), were purchased from Sigma-Aldrich, Inc. (St. Louis, MO,USA) (S1 Fig). Urethane (ethylcarbamate), lipopolysaccharide (LPS), basic fibroblast growth factor (bFGF), heparin, 12-O-tetradecanoylphorbol-13-acetate (TPA), Clodronate and Evans blue were purchased from Sigma Chemical Co. Bleomycin (BLM) from The antibodies used include: E-cadherin, N-cadherin, Vimentin, Nanog, Oct4, Snail1, CD133, Ki-67, cleaved-caspase 3, connexin 43 and fibronectin were obtained from BD Pharmingen. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody, Peroxidase substrate DAB (3′, 3′-diaminobenzidine) and AEC (3-amino-9-ethylcarbazole) were from Nichirei Bioscience (Tokyo, Japan). The mouse quantitative ELISA kits (TNF-α, MPO, ROS and 8-OHdG) were obtained from R&D Systems.

The procedure of western blotting was as described previously [49 (link)]. Tissues from the RA with or without MCT treatment were homogenized and lysed in a RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktails (Sigma). The protein concentration was determined with a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA, USA). Proteins were separated in 10% SDS-PAGE under reducing conditions and electrophoretically transferred onto an equilibrated polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK). Blots were probed with primary antibodies against connexin 43 (no. 610062; BD Biosciences, Oxford, UK), PKG (no. 3248; Cell Signaling, Beverly, MA, USA), ROCK1 (no. 4035; Cell Signaling, Beverly, MA, USA), Akt (no. 4685) (Cell Signaling, Beverly, MA, USA), phospho-Akt (no. 4060; Cell Signaling, Beverly, MA, USA), β-actin (no. ab6274; Abcam, Cambridge, MA, USA), and secondary antibodies conjugated with horseradish peroxidase. All bound antibodies were detected using an enhanced chemiluminescence detection system and analyzed with AlphaEaseFC software. All targeted bands were normalized to β-actin to confirm equal protein loading.

Cells were fixed in 4% PFA at 4 °C for 15 min and permeabilized with 0.1% Triton X-100 in PBS (Welgene, South Korea) for 5 min. After treatment with blocking solution containing 5% normal goat serum for 30 min, cells were stained with primary cardiac-specific antibodies against sarcomeric α-actin (Sigma) and connexin43 (BD Biosciences, Bedford, MA) at 4 °C overnight. The cells were washed three times with PBS and then stained with TRITC-conjugated secondary antibodies (Molecular Probes Inc., Eugene, OR, USA) at room temperature for 1 hour. After washing with PBS, nuclei were stained with DAPI (Invitrogen). All images were analyzed using LSM 510 and 710 META confocal microscopes (Carl Zeiss Inc., Oberkochen, Germany).