Ubi mcs 3flag sv40 puromycin

AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, PANC-1, and SW1990 cells were obtained from American Type Culture Collection (ATCC; USA), AsPC-1, BxPC-3, Capan-2, SW1990, and HPDE cell lines were cultured in RPMI 1640 (Gibco, USA), while Mia PaCa-2 and PANC-1 cell lines were grown in DMEM (Gibco, USA). Both medium were supplemented with 10%FBS and 1%Penicillin/Streptomycin. All cell lines were cultured in a humidified atmosphere containing 5%CO2/95% air at 37°C. All the cell lines had been authenticated through STR profiling and confirmed to be mycoplasma-free.
Lipofectamine3000 was purchased from Invitrogen (Invitrogen, USA); 3 small interfering RNAs (siRNA) (st-h-MINDY2-1 GCACAAGCCTCTCCATCAA, st-h-MINDY2-2 GCTGAGCAGTTTCTAAATA, and st-h-MINDY2-3 GTTCGAGTGTTTGAATATA) were provided by RiboBio (China). GeneChem (China) was responsible for designing and manufacturing lentivirus carrying negative control, MINDY2 overexpression vector (Ubi-MCS-3FLAG-SV40-puromycin), MINDY2-encoding short hairpin RNA (shRNA)(hU6-MCS-CMV-Puromycin), and shRNA targeting ACTN4(hU6-MCS-CMV-Puromycin). The directions were strictly followed during every infection or transfection step.

The following plasmids were purchased from GeneChem (Shanghai, China): lentivirus plasmid (Ubi-MCS-3FLAG-SV40-puromycin) carrying the full-length human STAMBP CDS (gene ID: 10617) and lentivirus plasmid (CMV-MCS-HA-SV40-Neomycin) carrying the full-length human RAI14 CDS (gene ID: 26064). Cells were plated at a density of 3 × 10⁵ cells/well and cultured for 24 h. Then, lentivirus was added to the cells at a multiplicity of infection of 10. After transfection for 48 h, 1 µg/ml puromycin (Selleck Chem, S7417) and 2 mg/ml G418 (Sigma, 108321-42-2) were used to select for cells with successfully transfected STAMBP and RAI14 plasmids, respectively.

Virus packaging was performed in HEK293T cells by co-transfection with lentiviral vectors with the packaging plasmid pHelper 1.0 vector (GeneChem Co., Ltd., Shanghai, China) and the envelope plasmid pHelper 2.0 vector (GeneChem Co., Ltd.) using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, supernatants containing lentiviral particles were collected, and the virus titer was quantified according to the manufacturer’s instructions. Lentiviral vectors encoding short hairpin RNAs (shRNAs) (Sh-1: ATTATTGTAACCACCCGTT; Sh-2 TGAGCAGTATTCCGAGAAA; Sh-3: CAATCATTTCATTGATCTT) targeting OGT were generated using the GV344 vector (hU6-MCS-Ubiquitin-Luc_firefly IRES-puromycin, GeneChem Co., Ltd., Shanghai, China). A scrambled GV344 vector (TTCTCCGAACGTGTCACGT) was used as the negative control. Stable transfectants overexpressing OGT were generated by lentiviral transduction using a GV341 vector (Ubi-MCS-3FLAG-SV40-puromycin, GeneChem Co., Ltd.). An empty vector was used as the negative control. Stable transfectants overexpressing EZH2 were generated by lentiviral transduction using a GV367 vector (Ubi-MCS-SV40-EGFP-IRES-puromycin, GeneChem Co., Ltd.). An empty vector was used as the negative control.