Genjet in vitro dna transfection reagent ver 2

293T cells were seeded at 5x10⁴ cells per well in 24-well plates in 0.5 ml D10 medium. Cells were co-transfected the following day with a full-length SIV_mac239Δnef clone (100 ng), pcDNA3.1-rBST-2 (50 ng) and either pCGCG, pCGCG-239-Nef or pCGCG-239-Nef mutants (100 ng). Differences in the amount of pcDNA3.1-rBST-2 were offset by the addition of empty pcDNA3.1 vector. Transfections were performed in duplicate using GenJet In Vitro DNA Transfection Reagent (Ver. II) (SignaGen Laboratories) according to the manufacturer’s instructions. Forty-eight hours post-transfection, the amount of virus released into the cell culture supernatant was measured by SIV p27 antigen-captured ELISA (Advanced Bioscience Laboratories. Inc.). Virus release was calculated at the percentage of maximal p27 release relative to control transfections in the absence of tetherin as previously described [12 (link)].

HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. HepG2 cells were grown in MEM containing 10% fetal bovine serum and 1 mM sodium pyruvate. HepG2 cells that were stably transfected with pMyc-DDB2 or control vector pcDNA3-Myc were selected in 400 µg/ml G418 (Sigma-Aldrich, MO, USA) and then maintained in the presence of 200 µg/ml G418. Transfection was carried out using Turbofect (Thermo, MA, USA) for HEK293T cells and GenJet™ In Vitro DNA Transfection Reagent (Ver. II) (SignaGen, MD, USA) for HepG2 cells. For MG-132 (Calbiochem, MA, USA) treatment, after 24 h of transfection, HEK293T cells were incubated with 10 μM MG-132 for an additional 24 h. HepG2 cells were starved overnight with serum-free medium and then treated with MG-132 or insulin (Sigma-Aldrich) at specific concentrations and durations. In another experiment, cells were treated with 10 μM MLN4924 for 24 h (MLN4924 was provided by Dr. Kuo-How Huang, National Taiwan University, Taiwan).

HepG2 cells were cotransfected with pHBI together with pGL3 (Promega) to normalize the transfection efficiency and an shRNA-expressing plasmid using GenJet In Vitro DNA Transfection Reagent (Ver. II) (SignaGen Laboratories, Ijamsville, MD) according to the manufacturer’s instructions. At 7 days post-transfection, the culture supernatant was used to measure the level of HBV production by real-time PCR, and cell lysates were used to check the transfection efficiency by a Luciferase Assay System (Promega).

HEK293 and HeLa cells were purchased from ECACC (Porton Down, Salisbury, UK) and cultured in Ham’s DMEM/Nutrient Mixture F-12 supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The MEF (DR-Wildtype) mouse embryonic fibroblasts and NIH3T3 mouse Swiss NIH embryo cell lines were purchased from ATCC (Manassas, VA, USA) and ECACC and grown in DMEM-high glucose and MEM, respectively, with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cell lines were cultured in a humid atmosphere of 5% CO₂ at 37 °C and all aforementioned reagents were supplied by Merck Millipore (Burlington, MA, USA). The HEK293, HeLa, and NIH3T3 cells were transfected with DNA constructs using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer instructions, whereas the MEF cells were transfected with GenJet™ In Vitro DNA Transfection Reagent (Ver. II) (SignaGen Laboratories, Rockville, MD, USA) according to the protocol provided by the supplier. The ratio of DNA/transfection reagent was optimized for each cell line in order to obtain the best transfection efficiency in the 24-well plate format.

HEK-293T cells and a human liver carcinoma cell line, HepG2, were used to express the major HBV proteins P, S, C, and X. Cells were originally obtained from ATCC and maintained and cultured in our laboratory according to our previous protocol [12 (link)]. The HEK-293T cells were seeded in 6-well collagen-coated plates (2 × 10⁵ cells/well) and incubated overnight. Transient transfection was performed using 1 μg plasmid using TransIT-LT1 reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s guidelines. HepG2 cells were seeded similarly (5 × 10⁵ cells/well) and transfected using GenJet™ In Vitro DNA Transfection Reagent ver II (SignaGen Laboratories) according to the manufacturer’s guidelines. After 48 h, cells were collected for further experiments. Transfection for immunofluorescence staining was performed following our previous protocol [12 (link)].