Mlc 2v

After treatment, iCMs were washed twice with PBS and then fixed for 5 min at RT using a PFA-4%/sucrose-2% solution. Fixed cells were incubated for 10 min with glycine 0.1 M and then washed with PBS for 5 min. Cells were then permeabilized with 0.3% Triton X (Triton X detergent, Sigma-Aldrich) in PBS for 30 min, followed by a 5-min wash with PBS. Cells were then blocked with 2% bovine serum albumin (BSA) (Merck) for 1 h at RT. Subsequently, they were incubated with PBS containing 0.1% Tween, 1% BSA, and the primary antibody overnight at 4°C. To assess cardiac differentiation, cells were stained using antibodies against Sarcomeric Actin (α-S-Actin) (Abcam 9465), cardiac Troponin I (cTnI, AbCam 47003), cardiac Troponin T (cTnT) (13-11 Thermo Fisher Scientific), Myosin light chain 2 atrial (MLC2a, Synaptic System 311011), and Myosin light chain 2 ventricular (MLC2v, Proteintech 10906) to assess presence of DNA breaks, cells were stained using antibodies against γ-H2AX (9718 Cell Signaling), to assess cytotoxicity cells were stained with cleaved Caspase-3 Ab (Cell Signaling Technologies 9664). To assess senescence induction cells were stained with P16 and P21 (Proteintech 10355) Antibodies.

Immunocytochemistry was directly performed on a 24-well plate as described previously [5 (link),6 (link),7 (link)]. Cells were fixed with 2% paraformaldehyde for 15 min, and then permeabilized with permeabilization buffer (0.05% Triton-X in PBS) for 5 min three times at room temperature. After incubating with blocking buffer (universal blocking buffer, BiogeneX, cat# HK083-50K) for ~45 min at room temperature, cells were incubated with primary antibodies against Troponin I (goat polyclonal, Abcam, Cambridge, MA, USA, cat# 188877, 1:400 dilution), MLC-2v (rabbit polyclonal, Proteintech, Rosemont, IL, USA, cat# 10906-1-AP, 1:200 dilution), and MLC-2a (mouse monoclonal, Synaptic Systems, Goettingen, Germany, cat# 311 011, 1:400 dilution) overnight at 4 °C. Following three 5-min washes with permeabilization buffer, cells were incubated with respective Alexa fluorogenic secondary antibodies (Invitrogen, Waltham, MA, USA) at 1:400 dilution at room temperature for 1 h. Cells were washed again with permeabilization buffer for 5 min three times. During the third wash, DAPI solution at 300 nM (Invitrogen) was added at 1:100 concentration.

mESCs, mESC-CMs and hiPSC-CMs were plated on matrigel-coated cover glasses and were fixed in 4% paraformaldehyde. Cells were stained with primary antibodies against OCT4 (1:50 dilution, Santa Cruz), EGFP (1:200 dilution, Frontier Institute), α-Actinin (1:800 dilution, Sigma EA-53), Troponin I (1:50 dilution, Santa Cruz), MLC-2v (1:50 dilution, ProteinTech Group) and MLC-2a (1:50 dilution, Synaptic Systems). Secondary antibodies were FITC-conjugated rabbit anti-goat IgG antibody, TRITC-conjugated rabbit anti-mouse IgG antibody, TRITC-conjugated swine anti-rabbit IgG (1:20 dilution, DAKO), and Alexa Fluor 488 goat anti-mouse IgG (1:200 dilution, Molecular Probes). Nucleus staining was performed with Hoechst 33342 (1:2500 dilution, Molecular Probes). F-actin staining was performed with Rhodamine Phalloidin (1:200 dilution, Molecular Probes).