Fugene

BeWo, JAR, HTR-8/SVneo and HUVEC cells were incubated for 24 h with SARS-CoV-2 (MOI=0.4). Next, cells were washed in PBS, lysed, and RNA was isolated by the total RNA purification kit (Norgen Biotek company, Thorold, ON, Canada). JAR and HTR-8/SVneo cells were seeded at 1.2 x 10⁴ cells/well in a 96-well plate (CellCarrierUltra, Optical Plate, PerkinElmer). After 10 h, cells were transfected with: (a) pCMV-SPIKEDelta-V5 + PCMV-hACE2; (b) pCMV-SPIKEDelta-V5 + pcDNA3; (c) pCMV-GFP + pcDNA3. For each condition, a transfection mix was prepared with OPTIMEM (Gibco #31985062), FuGENE (Promega, #E2311) and both plasmids at the ratio of 50 μl OPTIMEM: 3,5 μl FuGENE: 1 μg total pDNA. Plasmids in each mix were added at 1:1 ratio. Each transfection mix was incubated at RT for 15 min, then 10 μl of mix was added to each well (200 ng of total DNA per well). The plate was incubated at 37°C under 5% CO₂ for 24 h.

HEK GP-293 or HEK 293T cells were seeded at 60% confluency to produce viral supernatant. Retroviral plasmids (empty pMSCV-puro and pMSCV-PKR) were co-transfected with the pVSVG envelope plasmid into HEK GP-293 cells, whereas the pSL50 containing the sgRNA targeting PKR was co-transfected with the pVSVG envelope plasmid and the psPAX2 packaging plasmid (Gift from Didier Trono, Addgene plasmid # 12260; RRID:Addgene_12260) into HEK 293T cells.² FuGENE (Promega, Madison, WI) was used as the transfection reagent at a ratio of 4:1 (FuGENE:DNA). Culture supernatants were harvested after 48 h, aliquoted, and stored at −80°C. The supernatants containing retroviral or lentiviral particles were supplemented with 10 μg/ml DEAE-Dextran (Sigma-Aldrich, St. Louis, MO) and used to transduce THP-1 cells. Cells were selected using 1 μg/ml puromycin and analyzed by western blot to verify overexpression or deletion of PKR protein.

Lentiviral particles were created using a second-generation system. Briefly, following the instruction of Fugene (Promega, Madison, WI, USA), the mixture of pLenti-plasmid, vesicular stomatitis virus-glycoprotein, delta-8.91, and Fugene was added in the 6-well plate, and 1 × 10⁶ HEK293T cells were seeded on top of the mixture. After overnight incubation, medium was changed with DMEM with 2.5% (v/v) serum and 1% antibiotics. After 48 h, viral particles were harvested and filtered through 0.45-μm filter (GE healthcare, Hatfield, UK). Cells were transduced by filtered virus supernatant with polybrene (8ug/ml) 6 h after seeding. The next day virus medium was changed with selection medium containing appropriate antibiotics.

HEK GP2-293 cells seeded at 50% confluency were co-transfected with retroviral vector pMSCV-FAK and the VSV-G envelope plasmid using FuGENE (Promega, Madison, WA) at a ratio of 4:1 (FuGENE : DNA). Culture supernatants were harvested after 48 h, aliquoted, and stored at -80°C. Different volumes of supernatants containing retroviral particles supplemented with 10 µg/ml DEAE-Dextran (Sigma-Aldrich, UK) were used to transduce THP-1 cells, after which the viability of transduced cells was examined. Cells were selected by puromycin and analyzed by western blot to verify overexpression of FAK protein.

FuGENE (Promega, Madison, WI, USA) was used to transfect cells at a ratio of 2:1 (ratio FuGENE to DNA), according to the manufacturer’s protocol. Lentiviral and retroviral particles were generated in HEK293FT and Phoenix-Eco cells, respectively. For transduction, 30,000 cells were seeded in six-well plates and incubated for 24 h with viral particles supplemented with 1 µg/mL polybrene (Sigma, St. Louis, MO, USA) to enhance transduction efficiency. Cells were then selected with 1 µg/mL puromycin (Calbiochem, San Diego, CA, USA), or 800 µg/mL neomycin (Thermo Fisher Scientific, Waltham, MA, USA) for 7 days. Single cell clones were generated using limiting dilution. Lentiviral STX18 shRNA and scrambled controls were obtained from Sigma. For reintroduction of STX18, an empty vector (Mscv.PG.Neo) and a plasmid carrying STX18 (Mscv.STX18) were used. For sequences of shRNA, see Supplementary Table 2.

For BRAF functional assays murine-specific retrovirus containing BRAF constructs was produced via transient transfection (FuGene^®, Promega) of HEK-293T cells (ATCC) with the construct of interest and the EcoPak viral packaging vector, according to standard procedures^{65 (link)}. Briefly, 48 h following transfection Ba/F3 cells were transduced with viral particles via spinfection/polybrene. For spinfection, 1.5 mL of virus particle containing supernatant from 293 T culture was mixed with ~2 × 10⁶ Ba/F3 cells in a total volume of 3 mL of media containing HEPES buffer and 2uL of polybrene. Cells and virus were then centrifuged at 2000×g at 30 °C for 90 min, after which they were allowed to rest at 37 °C, 5% CO₂ for an additional 90 min. Following the rest period, the cells were washed with PBS, centrifuged, and suspended in fresh media (RPMI 1640, 10% FBS). After 48 h of transduction Ba/F3 cells were counted via hemacytometer and trypan blue exclusion every 2 days. For SAMD9/SAMD9L functional assays 293 T cells were transiently transfected with constructs of interest (FuGene^®, Promega).