E cadherin

Equal quantities of cell extracts were separated on a 10% SDS-PAGE gel, transferred, and analyzed by the Western lightning plus-ECL detection system (Perkin Elmer, Inc., Waltham, MA, USA). Briefly, antibodies against NDRG1, NDRG2, NDRG3 (Invitrogen), Snail (Abcam, Cambridge, UK), cyclin E, E-cadherin, STAT3, N-cadherin, Vimentin (Abgent, San Diego, CA, USA), Slug, p44/42 MAPK, Phospho-p44/42 MAPK, SAPK/JNK, Phospho-SAPK/JNK, p38 MAPK, Phospho-p38 MAPK (Cell Signaling, Danvers, MA, USA), Phospho-STAT3 and β-actin (Millipore) were used and the protein bands were detected and quantified with the ChemiGenius Image Capture System (Syngene, Cambridge, UK) and the GeneTools Program of ChemiGenius (Syngene).

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF) and proteins (20–40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, Rochester, NY). Antibodies of ATM, p-ATM, JAK1, p-JAK1, JAK2, p-JAK2, STAT3, and p-STAT3, were from Gene Tex (Irvine, CA), Antibodies of E-cadherin, N-cadherin, Vimentin, Snail, Zeb1, Twist and VEGF were obtained from Abgent (San Diego, CA) and antibodies of PD-L1, GAPDH, were from Cell Signaling (Danvers, MA).