Cyclin d1

Proteins were extracted using RIPA buffer, and protein concentration was measured using BCA Kit (EnoGene, Nanjing, China). Proteins were separated by 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane (Bio-Rad, USA). The membranes were blocked with 5 % skimmed milk at room temperature for 2 h, and washed in TBS-Tween 20. Subsequently, the membranes were incubated with anti-ROR2 (Biovision, Cat. 6702-100), anti-Bax (EnoGene, Cat. E11-0132C), anti-Bak (EnoGene, Cat. E11-0131C), anti-Bcl-2 (EnoGene, Cat. E10-30077), anti-Bcl-xl (EnoGene, Cat. E90209), anti-mTOR (EnoGene, Cat. E11-7156B), anti-survivin 1 (Biorbyt, Cat. orb394299), anti-PI3K (Biorbyt Cat. orb137259), anti-AKT (bioss, Cat. bs-0115R-1), anti-pAKT (bioss, Cat. bs-12458R-1), anti-PDK1 (EnoGene, Cat. E10-30154), anti-p21 (Abcam, Cat. ab215971-p21), anti-Cyclin D1 (Abcam, Cat. ab185241 - Cyclin D1), and control anti-GAPDH (EnoGene, Cat. E12-052) antibodies at 4 °C overnight. After washing in TBS-Tween 20, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP, EnoGene) at 37 °C for 1 h. Protein bands were detected using ECL Western Blotting System (Millipore, MA, U.S.A.) and visualized using image analyzer (DKSH, USA). The immunoblot signal was quantitated with ImageJ software and the values were normalized to the GAPDH band density.

Equal amounts of protein samples were separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Bands were probed immunologically using anti-Six3 (Santa Cruz), GSK‐3β (EnoGene), Phospho‐GSK‐3β (Ser9) (EnoGene), β‐catenin (EnoGene), Phospho-β‐catenin (Ser37) (EnoGene), Slug (EnoGene), E‐cadherin (EnoGene), N‐cadherin (EnoGene), c-Myc (EnoGene), and cyclin D1 (EnoGene). GAPDH (Cell signaling) was probed as an internal reference. Signals were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer’s instructions.

The human colorectal cancer LoVo cells were purchased from ATCC (Manassas, VA, USA). LoVo cells were cultured in F12K medium supplemented with 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin, at 37°C, 5% CO₂, and high humidity. The sources of antibodies (Abs) were as follows: IL-6 was purchased from R&D (Minneapolis, MN, USA), p-STAT3 was purchased from Abcam (Cambridge, MA, USA), cleaved caspase-3 was purchased from Cell Signaling (Beverly, MA, USA), and STAT3, cyclin D1, GAPDH, and the HRP-labeled secondary antibodies were purchased from EnoGene (Nanjing, China). Carboplatin was purchased from MelonePharma (Dalian, Liaoning, China). Annexin-V-FITC apoptosis detection kit, DAB Substrate Kit, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime (Beyotime, Shanghai, China). IL-6 ELISA kit was from NeoBioscience (Shenzhen, Guangdong, China).