Talin

Cells plated in 4-well chamber slides coated with Fibronectin (5 µg/ml, BD Biosciences) were exposed to DZ-50 treatment (5 µM) for 12 hrs. Cells were then fixed in 100% (v/v) methanol, and after blocking at 4°C (5% NGS, 0.3% Triton X), were exposed to the primary antibody (4°C, overnight). The following specific antibodies were used: ILK-1 (Cell Signaling Technology), ZO-1 (Invitrogen), Claudin-11 (Santa Cruz Biotechnology), Snail (Cell Signaling Technology), Talin (Millipore). Cells were then incubated with fluorochrome-conjugated secondary antibody (Invitrogen) (2 hrs, room temperature) and subjected to confocal microscopy using an Olympus FV1000 Confocal Microscope v1.21. and software version FV10-ASW 3.1.

Paraffin-embedded tumor samples were sectioned (4 μm) prior to antigen retrieval using citrate buffer and trypsin as described previously [31 (link)]. Following antigen retrieval, 3% normal goat serum diluted in phosphate-buffered saline was used for blocking for 30 min. Primary antibodies including Flii (1:200, Santa Cruz Biotechnology, Sydney, Australia), Flap-1/LRRFIP1 (1:400, Bioss, Woburn, USA), PCNA (1:200, Santa Cruz Biotechnology, Sydney, Australia), K1 (1:200, Abcam, Sydney, Australia), K14 (1:100, Abcam, Sydney, Australia), β-catenin (1:200, Santa Cruz Biotechnology, Sydney, Australia), SOX9 (1:1000, Abcam, Sydney, Australia), BrdU (1:500, Sigma-Aldrich, Sydney, Australia), pH3(s28) (1:400, Abcam, Sydney, Australia), γ-tubulin (1:500, Abcam, Sydney, Australia), Talin (1:500, Millipore, Sydney, Australia) were diluted in blocking buffer and applied. Species-specific Alexa Fluor 488, 568 or 633-conjugated secondary antibodies (1:400, Invitrogen, Melbourne, Australia) were diluted in phosphate-buffered saline and applied for detection. For detection of actin, directly conjugated Oregon Green 488 Phalloidin (1:400, Thermo Fisher, Melbourne, Australia) antibody was used. Nuclear counterstain 4,6-diamidino-2-phenyindole (DAPI) (1:5000) was applied last.

The total protein samples were extracted according to the kit instructions (Whole Protein Extraction Kit, Beyotime, China). The protein was separated by SDS-PAGE (voltage of 80/120 V) and transferred to PVDF membranes at a voltage of 60 V for 3 h. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature, incubated with primary antibody: GAPDH (1:8000, Cell Signaling Technology, USA); β-Actin (1:1000, Bioworld, China); OPN (1:1000, Abcam, USA); RUNX2 (1:1000, Abcam, USA); FAK (1:500, Cell Signaling Technology, USA); Talin (1:1000, Millipore, USA); Vinculin (1:800, Sigma-Aldrich, USA); Paxillin (1:1000, Becton Dickinson and Company, USA); Zyxin (1:700, Affinity, USA); YAP (1:700, Cell Signaling Technology, USA); P-YAP (1:1000, Cell Signaling Technology, USA) overnight at 4 °C with gentle shaking, and incubated with appropriate secondary antibodies: horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (1:5000, Fdbio Science, China); horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (1:5000, Fdbio science, China) at room temperature for 1 h. Protein expression was visualized using an exposure instrument (Tanon 5500, Tanon, China). Quantification of western blot data was performed by Gel-pro software.