Connexin 43

Proteins were extracted using RIPA Lysis buffer, which volume was adapted for every tissue. Proteins extracted were quantified using Bio-Rad Protein Assay System (Bio-Rad Laboratory, Melville, NY). Protein lysates were boiled in Laemmli buffer, run on BIO-RAD Mini-Protean TGXTM (10–20% Ready Gel Tris-HCl Gel System, 12-well comb #456–1095 and 10-well comb #456–1094) for 90 min at 120 Volt, and transferred on BIO-RAD Trans-Blot Turbo Mini or Midi PVDF membranes (Mini PVDF Transfer Packs #170–4156 and Midi PVDF Transfer Packs #170–4159) by semi dry Transfer (Trans-Blot Turbo Transfer System BIO-RAD). Specific proteins were detected using the following primary antibodies: Connexin-43 (1:850) (Cell Signalling, #3512, RRID:AB_2294590), β-actin HRP-conjugated (1:5000) (Sigma-Aldrich, #2228, RRID:AB_476697). Antibodies were diluted in 5% milk and incubation was done overnight at 4°C. Following secondary antibodies were used: HRP-conjugated anti-rabbit (1:5000) (Dako, #P0448, RRID:AB_2617138). Membranes were then washed and protein expression was analysed by chemiluminescence (Western Lightning Plus-ECL Perkin Elmer), using Fusion-FX (Vilber).

For Western blot analysis, the cultured AC16 cells were homogenized on ice in 1% CHAPS extraction buffer (150 mM KCl, 50 mM HEPES, pH 7.4, 0.1% CHAPS) containing complete™ EDTA-free Protease Inhibitor (Roche Bioscience) and 0.1 mM Na₃VO₄ to inhibit phosphatases. The homogenates were rotated for 30 min at 4°C to ensure the extraction of membrane proteins. After centrifugation at 15,000 × g for 120 min, the supernatant was collected, and protein concentrations were measured with BCA protein assay reagent (Pierce). Equal amounts of lysate proteins were resolved on 4–12% Bis-Tris NuPAGE gels, followed by standard Western blotting with the antibodies specified below. Chemiluminescent signals were scanned, and integrated density values were calculated with a chemiluminescent imaging system (Alpha Innotech). The antibodies of HIS, DSG2, Connexin 43, and GAPDH were purchased from Cell Signaling Technology (USA).

For immunofluorescent staining, the slides were washed with PBS and then incubated for 1 h with a primary antibody against cTnI (1:100 dilution, Abcam, Cambridge, UK), c-kit (1:50 dilution; R&D Systems, Minneapolis, USA), HIF-1α (1:100 dilution; Abcam, Cambridge, UK), Ki67 (1:50 dilution; Dako, Santa Clara, CA, USA) connexin43 (1:100 dilution; Cell Signaling, Massachusetts, USA), and TUNEL (In Situ Cell Death Detection Kit, Fluorescein, Sigma-Aldrich, St. Louis, MO, USA). The slides were then washed and incubated with respective anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG secondary antibodies (1:100 dilution; Jackson Immunoresearch, Ely, Cambridgeshire, UK) for 1 h at 37 °C. The nuclei were counterstained with the DNA binding dye, known as 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA), at 1 µg/mL. The slides were mounted in a VECTASHIELD antifade mounting medium and cardiac spheroids sections were analyzed using confocal microscopy (TCS SP8, Leica Microsystems, Wetzlar, Germany) [32 (link),33 (link),34 (link)]. To evaluate cellular apoptosis and hypoxia, the centermost CS sections were obtained by cutting at least 20 sections of 5 µm each for CS with a diameter of 200 µm (the number of cut sections to reach the centermost was dependent on the CS diameter). The specific antibodies used are shown in Table 1.