N cadherin

β-actin (Sigma Aldrich mouse monoclonal anti-β-actin, A2228, 1:2000), 42 kDa. N-cadherin (BD Biosciences mouse monoclonal anti-N-cadherin, 610921, 1:2000) 120 kDa. E-cadherin (BD Biosciences mouse monoclonal anti-E-cadherin, 610181, 1:2000) 120 kDa. Vimentin (Sigma-Aldrich goat polyclonal anti-Vimentin, V4630, 1:1000) 58 kDa. S100A10(BD Biosciences mouse monoclonal anti-S100A10, 610070, 1:2000) 11 kDa. Annexin A2 (BD Biosciences mouse monoclonal anti-Annexin II, 610069, 1:2000) 36 kDa. GAPDH (Biochain mouse monoclonal anti-GAPDH, Y3322, 1:2000) 36 kDa. p-S6K (Cell signaling rabbit monoclonal anti-pS6K, 9205 S, 1:1000) 70 kDa. FOXC2 (Bethyl laboratories rabbit polyclonal anti-FOXC2, A302-383A, 1:1000) 65–70 kDa. PAI-1 (Cell signaling rabbit monoclonal anti-PAI-1 D9C4, 11907, 1:2000) 48 kDa. uPAR (Santa Cruz rabbit polyclonal anti-uPA H149, sc-10815, 1:300) 55 kDa. p-ERK (Erk1/2) (Cell signaling rabbit polyclonal anti-Erk1/2 (Thr202/Tyr204), 9101, 1:1000) 42, 44 kDa.

Equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% non-fat skim milk/TBST [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.1%Tween-20] at room temperature for 1 h. Thereafter, they were detected with primary antibodies at 4°C overnight. Then, they were blotted for 1 h at room temperature with the help of an appropriate secondary antibody. Thereafter, enhanced chemiluminescence detection reagents were used (Amersham Pharmacia Biotech, USA). The primary antibody PRRX1was purchased from Abcam(UK). Caspase-3, Bcl-2, andγ-H2AX were purchased from Epitomics(UK). E-cadherin, ZO-1, Vimentin, N-cadherin were purchased from Becton, Dickinson and Company(USA). GAPDH was purchased from BOSTER(China).

Cells were seeded in 24-well plates and grown to confluence which typically required 3∼5 days. Cells were carefully washed with PBS and fixed in fresh 4% paraformaldehyde (PFA)/PBS at pH 7.4 for 10∼20 min at room temperature. After washing twice with PBS, PFA-fixed cells were permeabilized for 5 min in 0.2% Triton X-100 (Sigma-Aldrich) in PBS. Fixed cells were blocked in 10% BSA in PBS for 1 h at room temperature. As in our previous experiments [12] (link), primary antibodies, including rabbit anti-Na-K ATPase α1 (1∶100 dilution; Merck Millipore, Billerica, MA, USA), rabbit anti-zona occludens-1 (ZO-1; 1∶100; Invitrogen, Life Technologies), phospho-connexin 43 (1∶100; Merck Millipore), and N-cadherin (1∶100; Becton Dickinson), were incubated overnight at 4°C. Cells were washed twice with PBS and then incubated with a 1∶1000 dilution of FITC-labeled secondary antibodies (eBioscience, San Diego, CA, USA) in blocking buffer for 1 h at room temperature. After washing three times in PBS, all cells were stained with the DAPI nuclear marker (1∶5000; Invitrogen, Life Technologies) for 20 min at room temperature. A fluorescent mounting solution was added, the fluorescence was visualized on a Nikon Eclipse E-800 fluorescence microscope (Tokyo, Japan), and images were obtained with a spot digital camera.