To evaluate YAP nuclear localization, MSCs at each time point were fixed in 4% paraformaldehyde for 30 minutes at 37°C and then permeabilized with 0.05% Triton X-100 in PBS supplemented with 320mM sucrose. Samples were then incubated with anti-YAP antibody in 1% BSA in PBS (1:200, sc-101199, Santa Cruz Bio, Dallas, Texas) overnight at 4°C, followed by incubation with Alexa-Fluor 546 goat anti-mouse secondary (1:200, A-11030, Molecular Probes, Grand Island, NY) at RT for 1 hour. To visualize cells, actin was stained with Alexa-Fluor 488 (1:1000, A12379, Molecular Probes, Grand Island, NY) for 30 minutes and 4′, 6-diamidino-2-phenylindole (DAPI, ProLong ® Gold antifade reagent with DAPI, P36935, Molecular Probes, Grand Island, NY) to stain nuclei. Using a fluorescence microscope with a 100× objective (Zeiss Axioplan-2 fluorescent microscope, Jena, Germany), images were taken and the average YAP staining intensity and localization were quantified using ImageJ (with nuclear staining intensity normalized to cytoplasmic staining) as in 22 (link),28 (link). To investigate the effect of acto-myosin contractility on YAP nuclear localization, MSCs cultured on TCP through P2 were treated with Y27632 (Y27, 10 μM, for 1 h, EMD Millipore, Bedford, MA) for 1 hour prior to fixation and imaging.