Phospho myosin light chain

The samples on the nanofabricated coverslip were fixed with ice-cold 4% paraformaldehyde for 20 min, washed two times with phosphate-buffered saline (PBS), and permeablized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, cultures were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against Vinculin (1:200, Sigma-Aldrich), E-cadherin (1:200, Cell Signaling, 3195), phospho-Myosin Light Chain (1:200, Cell Signaling, 3674), γ-tubulin (1:100, Abcam, ab11321), YAP (1:100, Cell Signaling, 4912 and Santa Cruz, sc-15407), active β-catenin (1:100, Millipore, 05-665), Vimentin (1:100, Abcam), Slug (1:100, Cell Signaling, 9585), Twist (1:100, Santa Cruz, sc-15393), Vimentin (1:100, Abcam, ab24525), and WT1 (1:100, Santa Cruz, sc-192) for 3 h at room temperature. After washing with secondary antibodies and Alexa Fluor 594 conjugated phalloidin (1:40, Molecular Probes) and Hoescht (Invitrogen), cultures were incubated for 1 h at room temperature. The slides were mounted with an anti-fade reagent (SlowFade gold, Invitrogen) and taken by an inverted microscope (Zeiss Axiovert 200 M) with an X40 oil immersion objective (Zeiss, 1.6 NA).

hESCs were fixed in 3.7% formaldehyde for 10 minutes at room temperature (RT). They were then incubated for >30 minutes in a gelatin block containing 1X PBS (Invitrogen), fish gelatin (Sigma), normal goat serum (NGS) (Jackson), normal donkey serum (NDS) (Jackson), 0.1% bovine serum albumin (BSA) (Sigma) and 0.25% Triton X. NGS was removed when staining with goat antibodies. Blocked cells were then incubated in primary antibodies diluted in gelatin block as follows: SOX2 (1:100, StemGent), AP2α (1:200, Developmental Studies Hybridoma Bank), non-muscle myosin IIA (1:1,000, Cell Signaling), phospho-myosin light chain (1:200, Cell Signaling), OCT4 (1:300, Millipore), SSEA-3 (1:200, Millipore), Nanog (1:600, Cell Signaling), Brachyury (1:300, R&D Systems). Primary antibodies were incubated for either 1 hour at RT or overnight at 4 °C. Alexa Fluor secondary antibodies (Invitrogen) were incubated at a dilution of 1:250 for 45 minutes at RT in the dark. Phalloidin conjugated to Alexa Fluor 594 was incubated for 20 minutes at RT (1:40 in gelatin block, Invitrogen). Finally cells were either incubated with DAPI (1:1,000 in PBS, Pierce) and mounted to slides with ProLong Gold (Invitrogen), or mounted in ProLong Gold with DAPI (Invitrogen).

Cells were harvested for protein extraction and immunoblotting as described previously [14 (link)]. Primary antibodies used were for detection of β-Tubulin (#T9026, Sigma, St Louis, MO, USA), TSC1/hamartin (#370400, Life Technologies, Carlsbad, CA, USA), phospho-myosin light chain, (#3671, Cell Signaling, Danvers, MA, USA), and Rho-GTP (#BK036, Cytoskeleton, Inc Denver, CO, USA).