Anti twist

Anti-ZEB1 and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (CA, USA). The anti-E-cadherin, anti-vimentin, anti-snail, anti-slug, anti-twist and anti-p53 antibodies were purchased from Cell Signaling Technology (MA, USA). The anti-KLF5 antibody was purchased from Thermo Fisher (CA, USA). The anti-ZEB2 antibody was purchased from Proteintech (Rosemont, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (USA). KLF5 siRNA (siRNA ID stQ0005721-1), p53 siRNA (siRNA ID stB0002017C-1-5) and control siRNA were purchased from RiboBio (Guangzhou, China) and were used according to the manufacturer’s instructions.

Tissue specimens and cells were lysed in RIPA buffer (Beyotime, China) and the protein concentrations were measured by a BCA kit (Beyotime, China). Equal amounts of proteins were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After washing and blocking with sealing fluid, all membranes were incubated with rabbit polyclonal anti-RIOK1 (1: 2,000 dilution, Abcam, Cambridge, UK), anti-Cyclin B1 (1:1,000 dilution, Cell Signaling Technology, #4135), anti-p-AKT Ser473 (1:1,000 dilution, Cell Signaling Technology, #4060), anti-AKT (1:1,000 dilution, Cell Signaling Technology, #4691), anti-MMP2 (1:1,000 dilution, Cell Signaling Technology, #40994), anti-N-cadherin (1:1,000 dilution, Cell Signaling Technology, #13116), anti-vimentin (1:1,000 dilution, Cell Signaling Technology, #5741), anti-Cleaved PARP (1:1,000 dilution, Cell Signaling Technology, #5625), anti-Cleaved Caspase-3 (1:1,000 dilution, Cell Signaling Technology, #9664), anti-stat3 (1:1,000 dilution, Cell Signaling Technology, #12640), anti-p-stat3 Tyr705 (1:1,000 dilution, Cell Signaling Technology, #9145), anti-twist (1:1,000 dilution, Cell Signaling Technology, #69366), anti-E-cadherin (1:1,000 dilution, Abcam, Cambridge, UK), Tubulin (1:1,000 dilution, Abcam, Cambridge, UK). And then the corresponding secondary antibodies were incubated. ECL kit was used for detection.

The following antibodies and reagents were used: anti-OGN (Western blot) from R&D Systems anti-human OGN (IHC) from Sigma-Aldrich; anti-Akt, anti-phospho-Akt (Ser473), anti-phospho-EGFR (Y1068), anti-EGFR, anti-Slug, anti-Erk1/2, anti-Zeb-1, anti-Snail, anti-Twist, anti-Snail from Cell Signaling Technology; anti-CD31, anti-β-actin from prteintech; cross-linking reagent BS3 from Thermo Scientific Pierce; Eps15, epsin1 from Santa Cruz Biotechnology; AKT activator: sc79 from Selleck; OGN human enzyme-linked immunosorbent assay (ELISA) kit from USCN. All chemicals, unless otherwise specified, were purchased from Sigma-Aldrich.

The whole-cell extracts were lysed in RIPA buffer with a Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The Laemmli’s sample buffer was added to the lysates and boiled at 95 °C for 5 min then analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blotting was performed with anti-GPNMB, anti-vimentin, anti-Twist, anti-MMP2 and anti-GAPDH antibodies (Cell Signaling Technology, Danvers, MA, USA). Quantification of protein levels was analyzed by ImageJ software.

The sources of the antibodies and reagents used in this study were as follows: anti-CAV2 (Novus, NBP1-31116, USA), anti-GAPDH (Proteintech, 60004-1-Ig, China), anti-S100A2 (ImmunoWay, YN1118, China), anti-S100A4 (Bimake, A5363, USA), anti-S100A6 (Absin, abs137471, China), anti-S100A7 (Absin, abs139303, China), anti-S100A10 (Bimake, A5891, USA), anti-S100A11 (Affinity, DF7368, China), anti-S100A14 (Proteintech,10489-1-AP, China), anti-S100A16 (Affinity, DF4353, China), anti-S100P (Absin, abs137769, China), anti-E-Cadherin (Proteintech, 20874-1-AP, China), anti-N-Cadherin (Cell Signaling Technology, #13116, USA), anti-Twist (Cell Signaling Technology, #69366, USA), normal rabbit IgG (Cell Signaling Technology, #2729, USA), mouse IgG (Proteintech, B900620, China), and anti-Vimentin (Proteintech, 10366-1-AP, China). The secondary antibodies were HRP-conjugated Affinipure goat anti-rabbit IgG (Proteintech, SA00001-2, China) and HRP-conjugated Affinipure goat anti-mouse IgG (Proteintech, SA00001-1, China). MG132 (MedChemExpress, HY-13259, USA) was purchased from MedChemExpress. CHX (Beyotime, SC0353, China) was purchased from Beyotime.