Cd73 sirna

K-1 and SNU-790 cells (2 × 10⁵) were seeded onto 60 mm dishes and incubated in antibiotic-free culture medium for 24 h at 37 °C. The cell culture medium was replaced with antibiotic-free media containing 100 µM APCP, a CD73 inhibitor (Sigma-Aldrich, St. Louis, MO, USA; No. M3763), and cultured for 24 h (K-1 cells) and 48 h (SNU-790 cells), respectively.
CD73 knockdown was conducted using a CD73 siRNA (Dharmacon, Lafayette, CO, USA; No. D-008217-01) according to the manufacturer’s instructions. The target sequence of the siRNA used was as follows: CD73-siRNA, 5′-GAACCUGGCUGCUGUAUUG-3′, and that of non-targeting siRNA was 5′-UGGUUUACAUGUCGACUAA-3′. The cells were transfected with 20 nM non-targeting siRNA or CD73 siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA; No. 11668-019).

1, 2-Distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE), 1, 2-dioleoyl-sn-glycerol 3 phosphoethanolamine (DOPE) and 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL, USA), Octyl glucoside (OG), cholesterol and RPMI 1640 were purchased from Sigma-Aldrich. The lipofectamine 2000 was purchased from Invitrogen (USA). PE-conjugated anti-mouse CD73 (Cat No: 127205) and isotype-matched control monoclonal antibodies (mAbs) were purchased from Biolegend (USA). Phorbol 12-myristate 13-acetate (PMA)/ionomycin cocktail, Anti-mouse CD3-APC, anti-CD8a-PE-cy5, anti CD4-PE-cy5, CD25-FITC antibodies, anti-IFN-γ-FITC and antiIL-4-PE antibodies were purchased from BD Biosciences (San Diego, USA). CD73 siRNA and non-targeting siRNA (Cat No: D-001210-01-20) were obtained from Dharmacon (Thermo Fisher, Hilden, Germany). GE11 peptide (CGGGYHWYGYTPQNVI) with 1857 Da molecular weight and purity of 92.17% was purchased from China Peptides Co. (Shanghai, China). Liposomal Doxorubicin was purchased from Exir Nano Sina Company (Tehran, Iran). DEPC-treated water was applied in all experiments for siRNA containing samples. All other reagents were chemical grade.