Amershan imager 600

hiPSC-CMs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (all from Sigma-Aldrich). Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were then separated on 10–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline. Primary antibodies used for western blotting were as follows: anti-tubulin (1/1000; T5168, Sigma-Aldrich), anti-caspase-3 (1/500; #9662, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (1/200; #9661, Cell Signaling Technology), anti-Bax (1/500; #5023, Cell Signaling Technology), anti-Bcl-2 (1/200; #4223, Cell Signaling Technology) and anti-phospho-Histone γ-H2AX (1/1000; 05-636-200, Merck Millipore). Secondary antibodies were anti-IgG rabbit (1/5000; P0448 Dako, Santa Clara, CA, USA) and anti-IgG mouse (1/5000; A9044, Sigma-Aldrich). Detection was carried out using peroxidase-conjugated antibodies and SuperSignal^TM West Femto (ThermoFisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with the ImageJ software (ver. 1.53t, NIH, Bethesda, MD, USA).

Cells were lysed in RIPA buffer (1% NP40, 0,5% deoxycholate, 0,1% sodium dodecyl sulfate in Tris-buffered saline (TBS)) (Sigma-Aldrich) supplemented with protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk powder in TBS. Primary antibodies used for western blotting were: anti-Smad2 (1/500, Abcam, Cambridge, UK, ab228765), anti-YAP (1/1000, Cell Signaling Technology, Danvers, MA, USA, D8H1X), anti-phospho-YAP (1/1000, Cell Signaling Technology, D9W2I) and anti-GAPDH (1/1000, Cell Signaling Technology, D16H11). Secondary antibody was anti-IgG rabbit (1/5000, Dako, Santa Clara, CA, USA, P0448). Detection was carried out using peroxidase-conjugated antibodies and SuperSignal^TM West Femto (Thermo Fisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with ImageJ software (NIH, Bethesda, MD, USA).

The cytokine profile of rat heart samples was assessed using the commercial Proteome Profiler Rat Cytokine Array kit (R&D Systems, Minneapolis, MN, USA). Cryopreserved heart samples were homogenized in a solution of PBS with cOomplete™ protease inhibitor (Sigma-Aldrich) and 1% Triton® X-100 and kept cold. Nitrocellulose membranes with the captured antibodies of interest were incubated in an Assay Solution for 1 h under agitation at room temperature. Simultaneously, the homogenized samples were incubated with 15 µL of the detection antibody cocktail at room temperature for 1 h. Subsequently, the sample/antibody mixtures were added to the membranes and allowed to incubate overnight at 4 °C shaking. The next day, the membranes were washed 3 times, for 10 min each, and incubated with 2 mL of a solution of Streptavidin-HRP in Assay Solution for 30 min at room temperature with shaking. The membranes were rewashed 3 times and developed with the reagent mix provided by the kit. Images were acquired on an Amershan Imager 600 (GE Healthcare, Piscataway, NJ, USA). Protein expression was determined by densitometry using ImageJ.