Pcdh lentivector

Colon cancer cell lines were grown under the following conditions: SW480 and SW620 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Fisher SH3008102) supplemented with 10 % fetal bovine serum (FBS; Atlas FP-0500-A) and 2 mM glutamine (Fisher MT-25-005-CI). HCT116 and DLD-1 were cultured in RPMI-1640 medium (Fisher MT15040CM) supplemented with 10 % FBS and 2 mM glutamine. Doxycycline-inducible DLD-1 cells were created by transfecting Tet-inducible dnLEF-1N into DLD-1 TR7 cells (a generous gift from M. van de Wetering and H. Clevers) as previously described [8 (link)]. The induction of dnLEF-1 was achieved through addition of 0.01 μg/ml doxycycline to the media. Lentiviral constructs were cloned via Cold Fusion (System Biosciences) by inserting the coding sequence for flag-tagged dnLEF-1N into pCDH lentivector (System Biosciences; SBI CD533A-2). See “Luciferase reporter plasmid cloning” section for details regarding TK and SLC16A1 reporters.

cDNAs of HLA-DRA allele, 21 HLA-DRB1 alleles, 10 HLA-DQA1 alleles, 11 HLA-DQB1 alleles, three HLA-DPA1 alleles, 11 HLA-DPB1 alleles were obtained from lymphoblastoid cell lines of donors (740902.50; Macherey Nagel, RT300M; Enzynomics). Because of the SNPs near start codon and stop codon, cDNAs were amplified using each primer with short consensus sequence at the coding region and the sequence at 5′ UTR, 3′ UTR with mixed bases without a change in reading frames, and cloned into pCDH lentivector (#CD523A-1; System Biosciences) without any tag using Gibson assembly (EZ015TL; Enzynomics). The sequences were verified frequently.

293T, ACHN and HK-2 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 2 mmol/L L-glutamine in a humidified atmosphere at 37°C with 5% CO2. 786-O, OS-RC-2 and CaKi-1 cells were cultured in RPMI-1640 supplemented with 10% FBS and 2 mmol/L L-glutamine. Oligos corresponding to the target sequences were annealed and cloned into the HpaI and XhoI sites of the pSicoR plasmid (Addgene). The following target regions were chosen: PVT1-1#, GCUUGGAGGCUGAGGAGUU; PVT1-2#, CCCAACAGGAGGACAGCUU. Two putative PVT1 target sites were cloned into the XhoI-NotI site of the dual luciferase Psi-CHECK2 plasmid (Promega) separately. PVT1 cDNA was PCR-amplified by KOD-Plus-Neo and subcloned into EcoRI and BamHI sites of pCDH lentivector (System Biosciences, USA). All plasmids were verified by sequencing. The primers for vectors construction were provided in Supplementary Table 1, and the details of cell transfection were described in Supplementary Materials.