Plenti c myc ddk ires puro lentiviral gene expression vector

The coding sequence of the FUT4 gene (NCBI RefSeq; CCDS8301.1) in melanoma cells was cloned into the pLenti-C-Myc-DDK-IRES-Puro lentiviral gene expression vector (OriGene) between AscI and XhoI restriction sites. Lentiviral particles were generated using HEK293T cells transfected with control (empty pLenti-C-Myc-DDK-IRES-Puro) or pLenti-C-Myc-DDK-IRES-Puro-FUT4 lentiviral vectors along with VSVG and Δ8.9 packaging vectors as previously described^{14 (link)}. WM793 and WM1366 melanoma cells were subsequently infected with lentivirus, followed by antibiotic selection (1.5 μg/ml puromycin (InvivoGen)). Using the same lentiviral production and infection methods, shRNA-encoding plasmids (pLKO.1 lentiviral vector) (MISSION shRNA, Sigma-Aldrich) were used to stably knock down human FUT4 and L1CAM genes in melanoma cells. Three shRNAs for each gene were used for lentiviral infection, after expression validation, the one with the most knockdown effect was utilized for functional assays.

BCAT1 cDNA transcript variant 5 (NM_001178094) contained in PCMV6-Entry vector (PS100001, Origene) was obtained from Origene (Rockville, MD, USA). Site-directed mutagenesis of the BCAT1-CXXC motif was performed using the Quikchange II Site-directed Mutagenesis Kit (Agilent, UK) and mutagenic primers (C338SF 5′-GGTACAGCCTGTGTTGTTAGCCCAGTTTCTGATATACTG-3′, C338SR 5′-CAGTATATCAGAAACTGGGCTAACAACACAGGCTGTACC-3′). Successful mutation was confirmed by capillary sequencing at the University of Birmingham DNA Sequencing Facility. BCAT1-wild type (WT) and CXXC motif mutant BCAT1-C338S were sub-cloned into a pLenti-C-Myc-DDK-IRES-Puro lentiviral gene expression vector (Origene, UK, see Supplemental Figure S1) and Lentiviral particles containing pLenti-BCAT1 vectors were generated using Lenti-vpak packaging system (Origene, MD, USA) in HEK293T cells. For lentiviral transduction 2 × 10⁵ cells were added to 1 mL of viral suspension and centrifuged at 900× g for 2 h at 32 °C. Stably transduced cells overexpressing BCAT1 constructs were selected for incubation with complete RPMI supplemented with 0.5 µg/mL Puromycin.