Twist2

Immunohistochemistry (IHC) staining was performed in accordance with standard protocols. IHC staining was assessed by scores based on the percentage of positive cells (0: < 5%, 1: 5%–25%, 2: 25%–50%, 3: 50%–75%, and 4: > 75%) multiplied by scores based on the intensity of staining, (0: colorless, 1: light yellow, 2: brown, 3: and dark brown), with scores of 6–12 considered high expression and 0–4 considered low expression. Primary antibodies used in IHC were purchased from the following sources TWIST2 (Proteintech, Chicago, USA) and THBS2 (ZENBIO, Chengdu, China).

Western blotting analysis was conducted as described previously in our study (16 (link)). Total cellular proteins were harvested in IP lysis buffer (Beyotime, Shanghai, China) with a cocktail of proteinase and phosphatase inhibitors (Bimake.cn) and were measured by a BCA protein assay kit (Thermo Fisher Scientific, USA). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membrane (Millipore, USA). Then the membranes were blocked with 5% skimmed milk in Tris-Buffered saline (TBS) for 1 hour at RT and incubated with primary antibodies overnight at 4°C followed by incubation with DyLight fluorescent dye labelled secondary antibodies for 1 hour at RT. Finally, immunoblot signals were detected using an Odyssey Imaging System (LI-COR Biosciences, USA). Primary antibodies used in this analysis were listed as follows: TRIM50 (1:100 dilution, Abcam, ab174880), E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), Snail1 (1:1000, Proteintech, 13099-1-AP), Snail2 (1:2000, Proteintech, 12129-1-AP), Twist1 (1:1000, Proteintech, 25465-1-AP), Twist2 (1:1000, Proteintech, 11752-1-AP), ZEB1 (1:1000, Proteintech, 21544-1-AP), ZEB2 (1:2000, Proteintech, 14026-1-AP), and β-actin (1:5000, MultiSciences, ab008).