293t cells line

293T cells lines were purchased from ATCC (No. CRL-11268, Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), penicillin (10,000 Unit/mL) and streptomycin (10,000 μg/mL), nonessential amino acids (0.1 mM), and l-glutamine (2 mM) at 37 °C in a 5% CO₂ incubator. Gene knockdown used RNA interference experiments and the plasmid encoded NPC2 shRNA (pLKO.1-shNPC2) and control lacZ shRNA (pLKO.1-shlacZ). For the overexpression experiments, the plasmids (pLKO_AS3w.eGFP.puro, and pLV-NPC2) were obtained from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). We used TurboFectTM Reagent (Fermentas, Hanover, MD, USA) for the co-transfected packaging plasmid-pCMV-DR8.91, VSV-G envelope expressing plasmid-pMD.G, and one of four lentiviral constructs (pLKO.1-shlacZ, pLKO.1-shNPC2, pLKO_AS3w.eGFP.puro, pLV-NPC2) in 293T cells. Supernatants with lentiviruses were collected. To produce stable cell lines, LX2 cells and HSC-T6 cells were infected with lentivirus in polybrene (8 μg/mL) medium. After 24 h infection, 1 μg/mL purumycin was added for selecting stable cells. Gene-transfected stable cells (HSC-T6 shlacZ, HSC-T6 shNPC2, LX2 eGFP, and LX2 NPC2) were grown in DMEM with 1% fetal bovine serum (FBS) and 1 μg/mL puromycin [24 (link)].

Myc-CaP, LNCaP, and 293T cells lines (ATCC) used in this study were verified to be mycoplasma-free (Biotool), and human cell lines were authenticated by short tandem repeat (STR) loci profiling (ATCC). 293T cells were grown in DMEM (Corning), Myc-CaP and LNCaP in RPMI (Gibco), all supplemented with 10% heat inactivated fetal bovine serum (FBS; Corning) and 1% Penicillin-Streptomycin (10,000 U/ml; Life Technologies). All cell culture was performed in a 37 °C 5% CO₂ incubator with phosphate buffered saline (PBS; VWR) and 0.25% trypsin-EDTA (Gibco).