Twist

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).

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 the blocking procedure, membranes were incubated with primary antibodies, HRP-conjugated secondary antibodies, and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, Rochester, NY). GAPDH, bcl-2, bcl-xL, Notch, Hhg, Wnt1, CD44, and IL-6R antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). CD133 antibody was from Miltenyi Biotec (San Diego, CA), and ALDH antibody was obtained from BD Biosciences (San Jose, CA). p-Stat3, p-Akt, p-Erk, p-MEK, Oct4, Nanog, Sox2, and Mcl-1 antibodies were purchased from Cell Signaling (Danvers, MA). The antibodies of E-cad, N-cad, Twist, MMP9, and TGF-β1 were obtained from Abgent (San Diego, CA) and Integrin and VEGF antibodies were purchased from Abcam (Cambridge, UK).

Total cellular protein was isolated using 1% PMSF and RIPA lysis buffer (50 mM Tris–HCl pH 7.4, 150 m MNaCl, 1% NP-40, 0.1% SDS). Protein lysates were mixed with SDS-PAGE loading buffer and boiled at 100 °C for 5 min prior to electrophoresis. Samples were resolved on SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubation for 1 h with blocking buffer at room temperature, the membranes were incubated with rabbit anti-mouse polyclonal primary antibodies (GAPDH, SFRP1, Wnt, β-catenin, MMP2, PCNA, Twist, caspase3, MMP9, CDK1, NFAT, TGF, Bax, Bcl2 and Ap1; all from ABGENT, USA) at a dilution of 1:1000 overnight. Incubation with secondary antibodies was performed at room temperature for 1 h prior to detection with ECL reagent (Advansta, USA). The bands were obtained by GeneGnome 5 (Synoptics Ltd., UK).