Anti twist

IHC of all proteins was performed using the Novolink Max Polymer Detection System (Leica, Newcastle Upon Tyne, United Kingdom). The sections were deparaffinized in xylene, rehydrated in graded ethanol, and washed for 5 min with phosphate-buffered saline (PBS). The sections were boiled and immersed in Tris-EDTA (10 mM, pH 9.0) for 10 min at 125°C for antigen retrieval. Endogenous peroxidase activity was blocked for 30 minutes with 3% hydrogen peroxide in methanol. The sections were incubated with primary antibodies [anti-Snail, Abcam (ab53519), 1:100; anti-Twist, Santa Cruz (sc-15393), 1:50; anti-E-cad, BD Biosciences (610181), 1:200; anti-N-cad, Leica (NCL-L-N-Cad), 1:200; anti-Vimentin, SPRING (M3200), 1:400] overnight at 4°C in a humidified chamber after blocking. The sections were incubated with horseradish peroxidase-labeled secondary antibody for 10 minutes at room temperature after washing in PBS and counterstained with hematoxylin [16 ].

Cells were lysed in lysis buffer. Equal amounts of total proteins were loaded onto 4-12% SDS–PAGE gels and transferred to PVDF membranes (GE Healthcare Life Sciences, NJ, USA). The membranes were blocked with 5% milk dissolved in TBS containing 0.02% Tween 20 and incubated overnight at 4°C with specific primary antibodies. The membranes were subsequently incubated with specific horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a Fusion FX5 system ((Vilber Lourmat, France). The following primary antibodies were used: anti-cleaved caspase 3, anti-E-cadherin, anti-cleaved caspase 9, anti-cleaved PARP1 (Cell Signaling Technology), anti-SNAIL1, anti-Vimentin, anti-Twist, anti-Slug, anti-Zeb1, anti-Nanog, anti-Sox2, anti-CD44 (Santa Cruz), anti-N-cadherin and anti-Oct4 (BD Biosciences), anti-Survivin, anti-CD133 (Abcam), anti-ALDH (Avivasysbio), and anti-β-actin (Sigma).

Cell lysates (20 μg protein each) were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk and incubated with anti-phospho-p70^S6K (Thr389) (1:1,000) (Cell Signaling), anti-Twist (1:1,000) (Santa Cruz), anti-Sox9 (1:1,000) (Abcam), anti-N-cadherin (1:1,000) (Zymed), anti-E-cadherin (1:1,000) (BD Transduction Laboratories) in phosphate-buffered saline containing 0.1% Tween 20 rotating at 4°C overnight. After washing, the membrane was further incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h and developed with an enhanced chemiluminescent detection kit (Amersham).

Immunoblot assays were performed as described previously [62 (link)]. Primary antibodies were used as followed: anti-E-cadherin (610182, BD, San Jose, CA, USA), anti-α-catenin (610193, BD), anti-N-cadherin (610920, BD), anti-Vimentin (MS-129-P0, Thermo Scientific, Cheshire, UK), anti-Twist (sc-15393, Santa Cruz, Santa Cruz, CA, USA), anti-Snail (3895, Cell Signaling, Danvers, MA, USA), anti-Slug (AP2053a, Abgent, San Diego, CA, USA), anti-MMP2 (#4022, Cell Signaling), anti-MMP9 (#2551-1, Epitomics, Burlingame, CA, USA), anti-phospho-mTOR (Ser2448) (#5536, Cell Signaling), anti-mTOR (#2972, Cell Signaling), anti-phospho-IGF1 Receptor β (Tyr980) (#4568, Cell Signaling), anti-IGF1 Receptor β (#3018, Cell Signaling), anti-p21 (#2946, Cell Signaling), anti-p27 (#2552, Cell Signaling), anti-cyclin D1 (#2926, Cell Signaling), anti-cyclin E (sc-247, Santa Cruz), anti-β-actin (sc-1615, Santa Cruz) and anti-α-tubulin (MS-581-P0, Thermo Scientific). Protein levels were determined by measuring the intensity of bands on the blots using Image J (National Institutes of Health, Bethesda, Maryland, USA). Protein levels were normalized against an internal control β-actin or α-tubulin. The ratio was determined by dividing the normalized protein levels in expressing cells with that in control cells. The mean of ratio was obtained by averaging the ratios from several independent blots.

Cells were rinsed with ice-cold PBS and then lysed in RIPA buffer (Cell Signaling Technology) containing complete protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Roche Diagnostics), 5 mm dithiothreitol (DTT, Sigma), and 1 mm PMSF (Sigma) for 10 min on ice. Cells were then centrifuged at 15,000 × g for 10 min at 4 °C. Protein concentrations in the resulting supernatants were determined using the Bio-Rad protein assay (Bio-Rad) and samples were normalized for protein concentration. Aliquots containing 40 μg of total proteins each were loaded and separated by SDS-PAGE, then transferred to a PVDF membrane (Millipore) and stained by immunoblotting standard methods. The primary antibodies used for immunoblotting were anti-Scribble, anti-cleaved Caspase 3, anti-Slug, anti-Snail, anti-β-actin, anti-Hsp90, anti-PARP, anti-p38-MAPK, anti-phosphor-p38 MAPK, anti-eIF4E, anti-E-Cadherin, anti-N-Cadherin, anti-p-Akt (308), anti-Erk, anti-p-Erk, (1:1000; Cell Signaling Technology), anti-Twist, anti-HuR, and anti-Lamin B (1:1000, Santa Cruz).

Whole-cell protein extractions were prepared with a RIPA buffer (Beyotime, Beijing, China). The proteins were then electro-transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and blocked with 5% non-fat milk in Tris-buffered saline. Western blot analysis was performed with commercially available antibodies, anti-E-cadherin (Cell Signaling, MA), anti-Vimentin (Cell Signaling, MA), anti-caspase-3 (Cell Signaling, MA), anti-cleaved caspase-3 (Cell Signaling, MA), anti-Twist (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).