Itga6

Proteins were extracted using RIPA lysis buffer (ThermoFisher). Protein concentration was determined by the BCA Protein Assay Kit (Thermo Fisher, Waltham, MA). Proteins (40 µg) were run on 4–20% gradient gels and transferred onto PVDF membranes using a Trans-Blot Turbo transfer pack and Trans-Blo Turbo transfer system (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (LI-COR) and incubated with primary antibodies overnight at 4 °C. The primary antibodies were SNAI1 (1:500, 3895 s, Cell signaling), KLF17 (1:500, sc-398132, Santa Cruz), ITGA6 (1:500, sc-374057, Santa Cruz), MYC (1:500, 2272 s, Cell signaling), Cleaved PARP (1:500, 5625 s, Cell signaling), β-Actin (A-9) (1:500, sc-1616, Santa Cruz). p-AKT (1:500, #9217c, Cell signaling), p-ERK (1:500,), p-JNK (1:500, sc-6254, Santa Cruz). total-JNK (1:500,), ERK inhibitor U0126 (10 µM, 19–147, Millipore), JNK inhibitor SP 600,125 (10 µM, S1460, Selleckchem).

For protein extraction, cells were lysed in 50 μl of PRO-PREP Protein Extraction Solution (iNtRON Biotechnology; Seongnam-si, South Korea), homogenized using a 30-gauge needle, incubated for 30 min at 4 °C and then separated by centrifugation at 15 000 × g. After quantifying proteins in the extracts using the Bradford method, 20 μg protein was subjected to electrophoresis on 10% polyacrylamide gels in Tris/glycine (Invitrogen), transferred to a PVDF membrane (Millipore) and then probed with primary antibodies against LAMC2, ITGA6, ITGB4, EIF4E, VEGFA, DNMT3B, DNMT3L, and GAPDH (Santa Cruz), and DNMT3A (Abcam). Primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies (Invitrogen) and visualized using enhanced chemiluminescence reagents (Santa Cruz). The intensity of western bands were quantified using Image J. Each band was normalized with GAPDH and then presented as the relative intensity value of control- versus compressed sample.

Indomethacin, iRGD, and cilengitide were purchased from MCE (Junction, NJ, USA). MG132 and cyclohexamide (CHX) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). The ITGAV, fibronectin, ubiquitin, integrin β3 (ITGB3), p‐SMAD2 (S225), SMAD2, p‐SMAD3 (S423 and S425), and SMAD3 antibodies were obtained from Abcam (Cambridge, MA, USA). The p‐FAK (Tyr 925), FAK, PI3K p85α, p‐AKT (Ser 473), AKT, p‐GSK3β (S9), GSK3β, cyclin D1, CDK4, CDK6, SYVN1, NEDD4, ITCH, MYC, CBL, CBLB, anti‐Flag, and anti‐HA antibodies were obtained from CST (Beverly, MA, USA). The β‐Actin antibody was obtained from ZSGB (Beijing, China). The p‐PI3K p85α (Tyr 467), ITGA2, ITGA3, ITGA4, ITGA6, ITGA11, ITGB1, ITGB5, ITGB6, and ITGB8 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Ki67 antibody was obtained from Thermo Scientific (Waltham, MA, USA). Detailed information regarding the antibodies used within this study can be found in the Supplementary data. Plasmids encoding flag‐ITGAV and HA‐ubiquitin were obtained from Addgene (Watertown, MA, USA). Plasmids encoding HA‐SYVN1, MYC‐UB and MYC‐ITGB3 were from YouBio (Hunan, China). Lipofectamine 2000 was obtained from Invitrogen (Shanghai, China).