Anti twist1

The proteins were isolated through using SDS‐PAGE, and then the separated proteins were transferred onto PVDF membrane. The membrane was incubated with primary antibodies, anti‐KRT5 (1:2000, Proteintech, Chicago, USA), anti‐GAPDH (1:1000, Proteintech), anti‐Ki67 (1:1500, Proteintech), anti‐Twist1 (1:2000, Proteintech), and anti‐E‐cadherin (1:2000, Proteintech) at 4 °C overnight. The blots were exposed using enhanced chemiluminescence (ECL) system (Thermo Fisher).

Cell lysates were collected in RIPA lysis buffer (1% Triton-X-100, 20 mM Tris, pH 7.5, 137 mM NaCl, 1 mM EGTA, 10% glycerol, 1.5 mM MgCl₂, and protease inhibitor mixture and phosphatase inhibitors; latter 2 were from Roche). Lysates were sonicated and centrifuged at 4 °C. Per lane, whole-cell lysate was separated on 12% SDS-acrylamide gels and transferred on Immobilon PVDF membranes (Millipore). The membranes were probed with primary antibodies overnight at 4 °C and incubated for 1 h with secondary peroxidase-conjugated antibodies (CST). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and detected by the ChemiDoc XRS gel documentation system (Bio-rad). Antibodies include anti-Ube2v1 (Abcam, monoclonal ab151725), anti-E-cadherin (Santa Cruz, monoclonal sc-21791), anti-N-cadherin (Boster, polyclonal BA0673), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-Catenin (Cell Signaling Technology, monoclonal #8480), anti-Twist1 (Proteintech, 25465), anti-Snai1 (Bioss, bs-2441R), anti-LC3B (CST, monoclonal 3868), anti-SQSTM1/p62 (CST, polyclonal 5114), anti-H4K16ac (Immunoway, polyclonal YM3317), anti-Beclin1 (Boster, polyclonal PB0014), anti-histone H3 (Abcam, polyclonal ab1791), and anti-Sirt1 (Santa Cruz, monoclonal sc-74504).

The following antibodies were used in this study: anti-USP33, anti-slug, and anti-twist1 antibodies were purchased from ProteinTech Group (Chicago, IL, USA); anti-CXCR4 antibody was purchased from BD Bioscience (Franklin Lakes, NJ, USA); anti-HA and anti-Flag M1 antibodies were from Sigma-Aldrich (St. Louis, MO, United States); anti-ppERK, anti-ERK, and anti-β-arrestin2 antibodies were from Cell Signaling Technology (Boston, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-snai1, and anti-β-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA).
N-ethylmaleimide (NEM, deubiquitinase inhibitor), SDF-1 (SDF-1α, CXCR4 agonist), AMD3100 octahydrochloride hydrate (1,1′-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride, CXCR4 antagonist), dynasore hydrate (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide hydrate, dynamin inhibitor), HA affinity beads, and Flag affinity beads were all purchased from Sigma-Aldrich.

Cells were collected and lysed in RIPA buffer containing 1 mM PMSF (Beyotime, Cat. ST506, Shanghai, China). Protein samples were denatured at 100 °C for 10 min and then separated by 10% SDS-PAGE. After being transferred to a PVDF membrane (Millipore, Cat. IPVH00010, Bedford, USA), 5% skim milk in TBST was used to block non-specific binding for 2 h at room temperature. Then the membrane was incubated with the following primary antibodies: anti-CST2 (Cat. 19935-1-AP, 1:1000), anti-ISG15 (Cat. 15981-1-AP, 1:250), anti-E-cadherin (Cat. 20874-1-AP, 1:1000), anti-Twist1 (Cat. 13099-1-AP, 1:500), anti-Vimentin (Cat. 10366-1-AP, 1:10,000), and anti-GAPDH (Cat. 60004-1-Ig, 1:10000) (Proteintech, Wuhan, China) at 4 °C overnight. Next day, membranes were washed with TBST for three times and incubated with the secondary antibodies conjugated with horseradish peroxidase, goat anti-rabbit (Cat.70-GAR0072, 1:5000) or goat anti-mouse (Cat.70-GAM0072, 1:5000) (Lianke Multi Sciences, China), at room temperature for 1 h. ECL (Cat.20-500-120, Beit HaEmek, Israel) was used to detect the binding antibodies by ChemiDoc™ XRS⁺ System (Bio-Rad, USA).

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Wang et al., 2020 (link); Liu et al., 2021a (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-Twist1 (Proteintech, 25465-1), anti-Slug (Cell Signaling Technology, 9585), anti-Zeb1 (Cell Signaling Technology, 3396), anti-Snail (Cell Signaling Technology, 3879), anti-anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-598), anti-HDAC1 (Santa Cruz, sc-7872), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO₃), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.

Metformin, LY294002 and cycloheximide were obtained from Sigma-Aldrich (St. Louis, MO, USA). MG132 was purchased from Selleckchem (Houston, TX, USA). Recombinant human basic fibroblast growth factor (bFGF) was purchased from PeproTech (London, UK). The following antibodies were used in this study: anti-E-cadherin, anti-N-cadherin, anti-AKT, anti-phosphorylated-AKT were obtained from Abcam (Cambridge, MA, USA); anti-Vimentin, anti-GSK-3β, anti-phosphorylated-GSK-3β, anti-Twist1 and anti-GAPDH were obtained from Proteintech (Rosemont, IL, USA). Peroxidase-conjugated polyclonal goat anti-rabbit and polyclonal goat anti-mouse IgG antibodies were purchased from Thermo-Pierce (Rockford, IL, USA).