Anti slug

Western blotting analysis was performed using the same procedure demonstrated previously^{7 (link)}. Briefly, the radioimmunoprecipitation assay lysis buffer (Beyotime, China) was used to extract total cell protein, which was then quantified using the bicinchoninic acid protein assay kit (Beyotime, China) and resolved on a 6–12% sodium dodecyl sulphate-polyacrylamide gel, and then incubated with relevant primary antibodies. GAPDH and histone (ZSGBBIO, China) were used as loading controls. The following primary antibodies were utilised in this research: anti-PPARδ (Proteintech), anti-CXCR4 (Proteintech, China), anti-vimentin (Proteintech), anti-GAPDH (Proteintech), anti-β-catenin (Proteintech), anti-Slug (Proteintech) and anti-N-cadherin (Proteintech). We used GSK3787 (Abcam) and XAV-939 (Abcam, UK) as PPARδ and β-catenin inhibitors, respectively. The enhanced chemiluminescence system (Bio-Rad, Hercules, EDA USA) was used to detect protein expression levels and Scion imaging software was used to capture images.

Streptavidin-perosidase (SP) and diaminobenzidine (DAB) kits were purchased from Maixin Biotechnology Company (Fuzhou, China). The following primary antibodies were used for Western blot analysis: anti-CD36 (Santa Cruz, CA, USA), anti-E-cadherin, anti-TGF-β, anti-vimentin, anti-snail, anti-slug, anti-twist (Proteintech, IL, USA), and anti-GAPDH (Goodhere Biotechnology, Hangzhou, China).

Anti-PIMT (Abcam PLC, Cambridge, UK, 97446, 1:2000 dilution), anti-E-cadherin (Santa Cruz Biotechnology Inc., Santa Cruz, USA, sc-7870, 1:200 dilution), anti-vimentin (sc-7557, 1:250 dilution), anti-α tubulin (sc-5546, 1:400 dilution), anti-GRP78 (sc-1051, 1:200 dilution), anti-p53 (sc-1616, 1:200 dilution), anti-β actin (sc-32293, 1:200 dilution), anti-GAPDH (sc-25778), anti-Zeb1 (Atlas Antibodies AB, Stockholm, Sweden, HPA027524, 1:500 dilution), anti-Slug (sc-15391, 1:200 dilution), anti-Twist (Proteintech, Rosemont, IL, USA, 1:500 dilution), anti-Snail1 (Proteintech, 1:1000 dilution), anti-HIF1α (Novus Biologicals, Minneapolis, USA, NB100-479, 1:1000 dilution), and anti-HIF1α (Abcam, ab51608, 1:1000 dilution) antibodies were used in this study. Particularly, we used E-cadherin and vimentin to detect epithelial and mesenchymal properties in the cell lines. E-cadherin is a calcium-dependent transmembrane glycoprotein that mediates cell-cell adhesion in the polarized epithelium [42 (link)]. The loss of its expression is a hallmark of EMT [43 (link)]. Vimentin is a major marker of mesenchymal properties. Thapsigargin (Tg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Tunicamycin (Tn) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LW6, an inhibitor against HIF1α, was purchased from Merck Millipore (Darmstadt, Germany).