Dmrie c transfection reagent

An amount of 1 μg of linearized JFH1_T DNA plasmid was transcribed in vitro with the T7 RiboMAX Express Large kit (Promega) according to the supplied protocol. One million S29 cells were seeded in 10-cm culture dishes and allowed to adhere overnight. The following day, cells were washed twice with serum-free DMEM and then transfected for 3 h with 4 μl of the 10-μl RNA preparation using DMRIE-C transfection reagent (Invitrogen). Transfected cells were then washed once with complete medium and cultured at 37°C. For time course experiments, AO13 was added to culture fluids at indicated time points. At 48 h posttransfection, culture fluids were harvested and cells were trypsinized. Culture fluids and cell pellets were stored at −80°C for subsequent virus titrations and RT-PCR analysis.

Human T-ALL cell lines, namely, Jurkat, MOLT-4, and RPMI8402 were brought from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 37°C in 5% CO₂ in RPMI 1640 medium with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 10 mM Hepes, and 100 U of penicillin/streptomycin. Human HEK 293T cells used for lentivirus package were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 U of penicillin/streptomycin, 10 mM Hepes, and 2 mM L-glutamine (Invitrogen; Carlsbad, CA, USA) and were grown in humidified atmosphere with 5% CO₂ atmosphere at 37°C.
Lipofectamine™ 2000 Transfection Reagent was used for plasmids transfection in HEK293T cells. Delivery of siRNA targeting TRIM37 and the negative control (si-NC) was conducted using Invitrogen™ DMRIE-C Transfection Reagent.
The plasmids of pLVX-TRIM37-Puro (Clontech, Mountain Vie, CA, USA) or empty vector control, together with psPAX2 and pMD2.G (Addgen, Watertown, MA, USA), were delivered into HKE293T cells by Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) to generate lentivirus. Supernatants containing virus were harvested at 48h post-transfection. MOLT-4 cells were incubated with viral supernatants for 24h and then refreshed the supernatants with fresh medium supplemented with 0.25 μg/ml of puromycin for TRIM37-positive cell selection.

The cDNA encoding extracellular region of mouse CD155 was subcloned into the p3×FLAG-CMV-13 expression vector (Sigma-Aldrich) and transduced into MethA cells to generate transfectant stably expressing Flag-tagged sCD155 using DMRIE-C transfection reagent (Invitrogen). The transfectant was selected by G418 (Sigma-Aldrich) and passaged in the peritoneal cavity of mice.

An amount of 1 μg of linearized JFH1_T DNA plasmid was transcribed in vitro with the T7 RiboMAX Express Large kit (Promega) according to the supplied protocol. One million S29 cells were seeded in 10-cm culture dishes and allowed to adhere overnight. The following day, cells were washed twice with serum-free DMEM and then transfected for 3 h with 4 μl of the 10-μl RNA preparation using DMRIE-C transfection reagent (Invitrogen). Transfected cells were then washed once with complete medium and cultured at 37°C. For time course experiments, AO13 was added to culture fluids at indicated time points. At 48 h posttransfection, culture fluids were harvested and cells were trypsinized. Culture fluids and cell pellets were stored at −80°C for subsequent virus titrations and RT-PCR analysis.