Phospho faktyr397

Cells on fibers were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X100 solution and blocked in 5% goat serum. Paxillin (Tyr31) staining was done using primary rabbit anti-Paxillin antibodies (Invitrogen) at a dilution of 1:250 and incubated at 4 °C for 1 h. Secondary goat anti-rabbit Alexa Fluor 488 (Invitrogen) antibodies were incubated for 45 min at room temperature in the dark. For double immunostaining, total Paxillin (Host: Mouse, Invitrogen) primary antibodies at a dilution of 1:40 and Phospho-FAK (Tyr397) (Host: Rabbit, Invitrogen) primary antibodies at a dilution of 1:200 were used and incubated for 4 h. Secondary antibodies goat anti-mouse Alexa Fluor 647 and goat anti-rabbit Alexa Fluor 488 were used at dilution of 1:150 and incubated for 1.5 h at room temperature. F-Actin stress fibers were stained using Rhodamine Phalloidin (SantaCruz Biotechnologies). Cell nuclei were stained with 300 nM of DAPI (Invitrogen) for 5 min. The scaffolds were kept in 2 ml antifade imaging solution during imaging. Fluorescent images were taken using an Axio Observer Z1 microscope (Carl Zeiss). Actin live cell imaging was performed as per the manufacturer’s instructions on using the reagent CellLight Actin-RFP, Bacman 2.0 (Invitrogen).

Cultured cells (or cytospins with OCSC-enriched spheres) were fixed with 4% paraformaldehyde for 5 min at room temperature and then permeabilized in ice-cold PBS, 0.5% Triton X-100 for 3 min at 4 °C. After blocking for 1 h at room temperature with blocking buffer (PBS, 0.2% BSA, 1% donkey serum, 0.05% Tween-20 and 0.02% NaN3), cells were incubated for 2 h with the following primary antibodies, diluted in blocking buffer: FOXM1 (Cell Signaling, cat# 5436), FAK (Invitrogen, cat# 39-6500), phospho-FAK (Tyr397) (Invitrogen, cat# 700255), YAP (Cell Signaling, cat# 14074), WT1 (Abcam, cat# ab89901), αSMA (Abcam, cat# ab7817). Cells were then washed with PBS and incubated with the Alexa Fluor-conjugated secondary antibodies (Jackson Laboratories) for 1 h at room temperature. Nuclei were counterstained with DAPI (Sigma-Aldrich, cat# 32670). Images were acquired using the Leica SP8 Confocal microscope.