Lamin a c 4c11

To immunostain iPSCMs, cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 min, permeabilized with 0.1% Triton X-100 (Wako) for 5 min, and blocked with PBS containing 5% goat serum (Wako) for 1 hour at room temperature. After the samples were incubated with primary antibodies overnight at 4°C, they were incubated with Alexa Fluor–conjugated secondary antibodies for 1 hour at room temperature. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Images were captured using a confocal microscope (Carl Zeiss, LSM 880) and analyzed using ZEN (Carl Zeiss). The primary antibodies were Lamin A/C (4C11) (1:400; Cell Signaling Technologies, #4777), TNNT2 (13-11) (1:500; Thermo Fisher Scientific, #MA5-12960), and TEAD1 (1:100; Abcam, Cambridge, UK, #ab133533).

TC7 cells were grown in coverslides at a maximum of 80% confluence. They were fixed 10 min RT with 3.7% PFA in PBS 1X and washed 3 times with PBS 1X. All samples were sequentially treated with 0.1 M glycine in PBS 1X for 15 min, and 0.5% Triton X-100 in PBS 1X for 15 min RT. Primary antibodies were then incubated with overnight at 4 °C, washed with 0.05% Tween-20 in PBS, incubated for 1 h in the specific secondary antibody conjugated with Alexa 488 or Cy3 (Jackson, Ely, UK), 15 min with DAPI (1μg/mL), washed in PBS, and mounted with the antifading medium VECTA- SHIELD^® (Vector laboratories). Anti-HP1γ (2MOD-IG6, Thermo Scientific, Illkirch-Graffenstaden, France), laminB1 (ab65986, Abcam, Cambridge, UK), Lamin A/C (4C11, Cell Signaling, Danvers, MA, USA), and histone H2AX phospho-Ser139 (Millipore-Upstate 05–636, MA, USA) were used as primary antibodies. Nuclei were stained using 4, 6-diamidino-2-phenylindole (DAPI, 62248, Thermo Scientific Illkirch-Graffenstaden, France).
Microscopy images were obtained with a ZEISS Apotome.2 (Zeiss, Leipzig, Germany), a structured illumination microscope, using a 63× oil (1.4 NA) objective. To avoid overlapping signals, images were obtained by sequential excitation at 488 and 543 nm in order to detect A488 and Cy3, respectively. Images were processed using ZEISS ZEN lite software (Zeiss, Leipzig, Germany).