Cells were lysed using RIPA Lysis Buffer (Beyotime Institute of Biotechnology). The protein concentrations were determined using the bicinchoninic acid (BCA) assay. Proteins were separated via 12% SDS-PAGE and blocked with 5% bovine serum albumin at 4°C overnight. A total of 20 ml of 12% separation gel was prepared, and the 5% stacking gel was added on top of the separation gel. In total, 20–40 µl protein samples with 5 µl protein marker were added to the corresponding lanes. Then the protein samples were transferred to the cellulose nitrate membranes by electrophoresis. The cellulose nitrate membranes were incubated with the following primary antibodies at 4°C overnight: Anti-c-Myc (product code ab32072; 1:2,000), anti-cyclin D1 (product code ab16663; 1:2,000), anti-Slug (product code ab51772; 1:2,000), anti-Twist (product code ab50887; 1:2,000), anti-E-cadherin (product code ab40772; 1:2,000), anti-N-cadherin (product code ab76011; 1:2,000), anti-vimentin (product code ab92547; 1:2,000), anti-Sox2 (product code ab92494; 1:2,000), anti-Oct4 (product code ab200834; 1:2,000), anti-KLF4 (product code ab215036; 1:2,000), anti-ALDH1 (product code ab177463; 1:2,000), anti-CD44 (product code ab189524; 1:2,000), anti-phosphorylated (p)-β-catenin (product code ab81305; 1:2,000) and anti-RORβ antibody (product code ab228650; 1:1,000; all from Epitomics; Abcam), anti-Bcl-2 like protein 11 (BCL2L11; cat. no. AP8553c; 1:1,000; Abgent, Inc.) and anti-GAPDH (cat. no. KC-5G5; 1:5,000; KangChen Biotech Co., Ltd.). Then the cellulose nitrate membranes were incubated with the secondary antibodies at room temperature for 1 h: Goat anti-rabbit IgG H&L (HRP) (product code ab6721; 1:2,000) or goat anti-mouse IgG H&L (HRP) (product code ab6789; 1:2,000; both from Epitomics; Abcam). Protein bands were visualized with ECL reagent (PerkinElmer, Inc.), according to the manufacturer's protocol. BandScan software (V5.0; Glyko Biomedical, Ltd.) was used for densitometric analysis.

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