Ab82541

VSMCs were cultured on cover slides in 24‐well plates. After washing with PBS three times, they were fixed in 4% PFA for 15 minutes followed by permeabilization with 0.1% Triton X‐100 in PBS for half an hour. Cells were blocked by 10% BSA and then incubated with the following primary antibodies at 4°C overnight: anti‐calponin (1:100, Abcam, ab46794), anti‐α‐SMA (1:100, Abcam, ab5694), anti‐MYH11 (1:100, Abcam, ab82541), anti‐Smoothelin (1:100, Abcam, ab8969), p‐p53 (1:100, Abcam, ab33889) and anti‐ki‐67 (1:100, Abcam, ab15580). Next, cells were incubated in the dark with fluorescent‐labelled secondary antibodies (1:1000) for 1 hour at room temperature. Subsequently, VSMCs were washed with PBS three times and mounted with 4′, 6‐diamidino‐2‐phenylindole (DAPI). Finally, five randomly selected areas of each slide were photographed under a fluorescence microscope.

Cells were grown on round 14 mm glass coverslips (Thermo Scientific, Waltham, MA, USA), fixed in ice-cold methanol for 10 min at room temperature and then blocked in PBS/3% w/v BSA (A6003, Sigma-Aldrich, St. Louis, MO, USA) for 30 min and stained with primary antibody anti-smooth muscle Myosin heavy chain 11 (ab82541, Abcam, Cambridge, UK) for 1 h at room temperature. Cells were washed in PBS pH 7.45 and incubated with Alexa Fluor^® 488 goat anti-rabbit IgG (H + L) (A-11008, Invitrogen, Thermofisher, Carlsbad, CA, USA) secondary antibody for 45 min at room temperature in darkness and DNA was counterstained with 4′,6-diamino-2-fenilindol (DAPI). After three washes, coverslips were mounted in Fluoromount™ Aqueous Mounting Medium (F4680, Sigma-Aldrich, St. Louis, MO, USA). Image acquisition was performed with Leica TCS STED CW SP8 Super-Resolution Microscope with a 40× lens and recording optical sections every 0.3 µm. Image analysis was done analyzing MYH11 staining fluorescence of each cell per cell area in MIT and PLQ VSMCs with ImageJ software (National Institute of Health, Bethesda, MD, USA).