Dharmafect 4 transfection reagent

TrHBMEC (2 x 10⁵) were seeded into individual wells of a 6-well plate containing a single 18mm glass coverslips precoated with 0.2% gelatin in 2ml of complete medium and 24 h after seeding the medium was replaced with 2ml complete medium minus antibiotics for an additional 16h. On-target plus non-targeting siRNA (control) and on-target plus human NQO1siRNA smartpool were purchased from Dharmacon (Lafayette, CO). siRNAs and DharmaFect 4 transfection reagent (Dharmacon) were prepared for transfection (6-well plate) as described by the manufacturer (see: http://dharmacon.gelifesciences.com/uploadedFiles/Resources/basic-dharmafect-protocol.pdf)) For these studies each transfection was carried out using 50nM of siRNA in combination with 2μl of DharmaFect 4 transfection reagent. Cells were treated with siRNA/transfection reagent diluted in DMEM containing 10% FBS (no antibiotics) for 72 h after which the coverslips were removed and processed for immunocytochemistry as describe below.

For gene hit validation experiments, KO cell lines were generated using the CRISPR-Cas9 system. To generate bulk cell pools, HCT 116 Cas9 and Panc-1 Cas9 cells were transfected with two to three independent sgRNAs targeting ILKAP (see Table 2) using DharmaFECT 4 Transfection reagent (Horizon Discovery, T-2004–03). according to manufacturer’s instructions. After 2 days, cells were used for tumor killing assay and Simple Western analysis. Limiting dilution and clonal expansion was used to generate HCT 116 ILKAP KO monoclonal cell pools for further analysis. Gene disruptions were confirmed by sequence analysis and Simple Western analysis. To generate ICAM1 KO polyclonal cell pools, HCT 116 Cas9 and Panc-1 Cas9 were transfected with two independent sgRNAs targeting ICAM1 (see Table 3) using DharmaFECT 4 Transfection reagent (Horizon Discovery, T-2004–03) according to manufacturer’s instructions. UACC-257 cells were co-transfected with Cas9 protein and two independent sgRNAs targeting ICAM1 using Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo Fisher Scientific, CMAX00008) according to manufacturer’s instructions. ICAM-1 negative cells were sorted using fluorescence-activated cell sorting (FACS) and further expanded, then used for tumor killing assay and validation experiments. Depletion of ICAM1 was periodically checked by cell surface staining.

Transfection was performed using DharmaFECT 4 transfection reagent (GE Dharmacon, Lafayette, CO, USA) in a 96-well white plate with a clear bottom. A smart pool of non-targeting control (NTC) siRNA (D-001206-13; GE Dharmacon) and caspase 3 siRNA (L-004307-00; GE Dharmacon) were used as negative and positive control, respectively. siRNA was diluted in DharmaFECT cell culture reagent (GE Dharmacon) and used at a final concentration of 50 nM. Transfection reagent and siRNA were mixed and incubated at 25°C for 30 minutes to form siRNA-liposome complex. Huh7 cells at 1.5 x 10⁴ cells per well were allowed to plate onto the transfection mixture and were then incubated for 24 hours. The media were then aspirated out and the transfected cells were infected with supernatant containing DENV at MOI 10 and incubated for 48 hours. Cell viability and caspase 3 activity were measured as previously described.