Sc 166081

Dissociated CMs were fixed with 4% PFA and stained with Troponin T (cTnT, 1 : 2000, ab64623, Abcam, Cambridge, MA, USA), MYBPC (1 : 400, sc-166081, Santa Cruz Biotechnology), and TPM1 (1 : 200, sc-73225, Santa Cruz Biotechnology) primary antibodies, followed by labeling with secondary antibodies. Images were obtained with Olympus IX51 phase contrast microscope equipped with fluorescence optics and Olympus DP308W camera (Olympus Corporation) or with Zeiss AxioScope A1 fluorescent microscope and Zeiss AxioCam MRc5 camera (Carl Zeiss, Jena, Germany). Size of the Troponin T stained CMs was analyzed from 46 to 50 CMs in each cell line by in-house made software (unpublished method). CMs were analyzed from pictures obtained with Olympus IX51 phase contrast microscope. The proportion of multinucleated CMs was determined from the same images (46–50 CMs/cell line).

Dissociated CMs were fixed with 4% PFA and stained with troponin T (cTnT, 1:2000, ab64623, Abcam, Cambridge, USA), MYBPC (1:400, sc-166081, Santa Cruz Biotechnology, TX, USA), connexin 43 (Cx43, 1:1000, C6219, Merk), and MYH6 (1:500, R&D Systems, Minneapolis, USA) primary antibodies, followed by labeling with secondary antibodies: Alexa Fluor 488–conjugated anti-goat, anti-mouse, and anti-rabbit antibodies respectively (1:800, A11055, A21202, A21206, Thermo Fisher Scientific). Images were obtained with Nikon N-SIM (super-resolution system microscope equipped with EM CCD camera iXon3 DU-897E (Andor Technology Ltd, Belfast, UK)). In-house-built selective plane illumination microscopy (SPIM) fluorescent imaging system was used to create the 3D images of the reporter CM cluster (Vuornos et al. 2019 (link)).
Cells at day 15 of CM differentiation were fixed in 4% paraformaldehyde and permeabilized by FACS buffer with 0.1% Triton™ X-100. Troponin T antibody diluted in FACS buffer (1:100) as primary antibody and Alexa Fluor 647–conjugated anti-goat (1:800, Thermo Fisher Scientific) as secondary antibody were used for FACS analysis. The resulting data were analyzed using FlowJo v8.5.2.

hiPSC-derived CMs were lysed in M-PER protein extraction reagent (Thermo Scientific, Life Technologies Ltd.), supplemented with complete protease inhibitor cocktail (Roche Diagnostics). The protein concentration was quantified with BCA protein assay kit (Thermo Scientific, Life Technologies Ltd.). 10 μg of protein was run to 4–15% mini-PROTEAN TGX precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membrane (Amersham Hybond-P, GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% milk for 1 h at RT and proteins were stained with MYBPC (1 : 1500, sc-166081, Santa Cruz Biotechnology), cTnT (1 : 2000, ab64623, Abcam), TPM1 (1 : 200, sc-73225, Santa Cruz Biotechnology), or β-actin (1 : 1000, sc-47778, Santa Cruz Biotechnology) primary antibodies over night at +4°C. Horseradish peroxidase- (HRP-) conjugated polyclonal rabbit anti-mouse (DAKO, P0260) and rabbit anti-goat IgG (Santa Cruz Biotechnology, sc-2922) were used as secondary antibodies. Stained proteins were detected by using Clarity ECL substrate (Bio-Rad) and visualized by Molecular Imager ChemiDOc XRS+ (Bio-Rad). ImageJ software (National Institutes of Health, USA) was used to compare the expression of MYBPC, cTnT, and TPM1 with the β-actin expression from the same cell line.