Hrp conjugated goat anti mouse or anti rabbit secondary antibodies

Celecoxib, cisplatin, 5-fluorouracil (5-FU) and prostaglandin E₂ (PGE₂) were purchased from Sigma-Aldrich. Each compound was prepared as 1 mM stock solution in dimethylsulfoxide (DMSO) for dilution into various concentrations. The following mouse or rabbit monoclonal primary antibodies were used: anti-E-cadherin (Abcam), anti-MMP-2 (Abcam), anti-Vementin (Abcam), anti-c-MYC (Abcam), anti-Axin-2 (Abcam), anti-Cyclin-D1 (Abcam), anti- β-catenin (Cell Signaling Technology), anti-Survivin (Cell Signaling Technology), anti-SOX-2 (Cell Signaling Technology), anti-p-GSK-3β (Cell Signaling Technology), anti-GAPDH (Beyotime Biotechnology) and anti-Tubulin (Beyotime Biotechnology). HRP conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Beyotime Biotechnology.

HMVECs and TALL-104 cells were mixed (1:1) in culture for 12 h. Paraformaldehyde (4%) was added to the culture for 10 min at room temperature prior to cell lysate isolation, as described below.
For immunoblotting, cells were solubilized in ice-cold cell lysis buffer containing 6.52 mg/mL HEPES, 0.42 mg/mL NaF, 8.64 mg/mL NaCl, 0.2 mg/mL MgCl₂, 5% NP-40, and protease inhibitor cocktail (TOPSCIENCE, Shanghai, China). Proteins were fractionated by SDS-PAGE and transferred to PVDF membrane (Millipore, Burlington, MA, USA) and then blocked in 5% milk in PBST. Subsequently, the membrane was incubated overnight at 4 °C with one of a series of primary antibodies against mouse or rabbit PD-L1 (Cell Signaling, Danvers, MA, USA), PD-1 (Cell Signaling), VE-cad (Invitrogen, Waltham, MA, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Syndecan-1 (Santa Cruz Biotechnology), LEF1 (Cell Signaling), YKL-40 (our lab), CCR5 (Abcam, Cambridge, UK), AKT, pAKT (Cell Signaling), ERK, pERK, PI3K (Santa Cruz Biotechnology), Vimentin (Dako), SMa (Abcam), E-cad (Dako, MA, USA), GAPDH (Beyotime), or β-actin (Sigma-Aldrich). The membrane was then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Beyotime). The specific chemiluminescence signal was detected using the ECL reagent (ThermoFisher Scientific).