Prmi 1640 culture medium

Canine IL-15 cDNA (NCBI Reference Sequence: NM_001197188.1) was amplified and cloned into the pLAS3w vector. Canine IL-15 lentivirus was generated via transfection of 293T cell line with lentiviral transfer vector (pLAS3w-IL-15), pCMVΔR8.91 plasmid (expression of Gag, Pol, and Rev protein), and pMD.G plasmid (expression of VSV-G protein). All package plasmids (pLAS3w, pCMVΔR8.91, and pMD.G) were obtained from RNAi Core Laboratory in Academia Sinica, Taiwan. NK-92 cells were transduced with the IL-15 lentiviral particles, and cells with high expression were further screened by puromycin (Sigma-Aldrich, Taufkirchen, Germany) and sorted by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA). Transfected NK-92/IL-15 feeder cells were cultured in PRMI-1640 culture medium (Gibco, Grand Island, NY, USA), supplemented with 10% FBS (Invitrogen, Grand Island, NY, USA), 100 IU/mL IL-2 (Proleukin, Clinigen Healthcare, Staffordshire, UK), and 2 mM L-glutamine (Sigma-Aldrich, Taufkirchen, Germany) to maintain gene expression.

PSMA-617 (ABX Advanced Biochemical Compounds); WST-1 assay kit and Cytotoxicity detection kit (lactate dehydrogenase, LDH) (Roche); Propidium Iodide(PI) Reagent (Invitrogen); cresyl violet (Sigma-Aldrich); PRMI 1640 culture medium, HAM’-12 medium, foetal bovine serum (FBS) and penicillin/streptomycin solution (10,000 U/ml) (Gibco™); Cellytic™ MT Cell Lysis Reagent (Sigma-Aldrich); halt protease and phosphatase inhibitor and Pierce™ BCA protein assay kit (Thermo Scientific); Western Blotting Luminol Reagent (Santa Cruz Biotechnology); Immobilon-P polyvinylidene difluoride membrane (PVDF) (Millipore); high performance chemiluminescence film (Amersham International plc); Western Blot Stripping Buffer (Thermo Scientific). All other chemical substances, which are not explicitly mentioned, were purchased from Sigma-Aldrich or Merck.