Cells were fixed with 4% formaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS. Cells were stained with the primary and secondary antibodies for 1 hour each at room temperature. The following primary antibodies against Myosin-11 (Abcam), Calponin1 (Sigma-Aldrich), α-SMA (Sigma-Aldrich) and secondary antibodies either anti-Rabbit IgG (H+L), or anti-Mouse IgG (H+L) conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) were used. Nuclei were counterstained with DAPI. Percentage of cells positive for of of Myosin-11, Calponin 1 and α-SMA was obtained from three independent experiments. Two hundred of cells were counted in each experiment. Fluorescence and time-lapse images were obtained with an OLYMPUS microscope as described below and processed with Adobe Photoshop CS6 and Adobe Illustrator CS6 (Adobe Systems). carbachol-induced contractility assay was carried out as described previously19 (link), 20 (link). Briefly, Phase-contrast time lapse of carbachol (100 μM, Alfa Aesar) induced contraction of iSMC was recorded with a DP70 microscope digital camera with an IX71 phase-contrast fluorescent motorized inverted microscope using DP Controller and edited with DP Manager (Olympus) in a time-lapse manner at 1-minute intervals for 30min. Contraction was determined by tracking individual cells in the videos.