Trasfectin

To knockdown FL BARD1 gene expression, pGIPZ Lentiviral shRNAmir targeting human BARD1 were purchased from Open Biosystems (Thermo Fisher Scientific, Inc.). We used two different shRNA against FL BARD1: V2LHS_93186 and V3LHS_365581. A non-silencing pGIPZ Lentiviral shRNAmir was used as control (RHS4346). HEK293 were transfected with 10µg of shRNA plasmid DNA and 30µl of Trans-Lentiviral packaging Mix (OpenBiosystem) and 25µl TrasFectin (Bio-Rad) in 10mm plate. The supernatants (10 ml for points) were harvested after 24 hours, centrifuged at low speed to remove cell debris and filtered through a 0.45 μm filter 33 (link), 34 . In vitro transduction and determination of lentivector Titre was performer as already reported 35 (link). After 48 hours of incubation, the transduced cells were examined microscopically for the presence of TurboGFP expression (70-90%). To obtain 100% GFP positive cells we added puromycin in the medium for additional 10 days.

To knock-down HIF1A and EPAS1 expression, the pGIPZ lentiviral shRNAmir that targets human HIF1A and EPAS1 were purchased from Open Biosystems (Thermo Fisher Scientific, Inc.). We used two different shRNAs for each gene. The shRNAs against EPAS1 were: V2LHS113750 (RHS4430-98894439) and V2LHS-113750 (RHS4430-98851126). The shRNAs against HIF1A were: V2LHS_132152 (RHS4430-98513964) and V2LHS_236718 (RHS4430-98513880). A non-silencing pGIPZ lentiviral shRNAmir was used as the control (RHS4346).
HEK293T were transfected using 10 μg shRNA plasmid DNA, 30 μl Trans-Lentiviral Packaging Mix (OpenBiosystem), and 25 μl TrasFectin (Bio-Rad), in 10-mm plates. The supernatants (10 ml per condition) were harvested after 24 h, centrifuged at a low speed to remove cell debris, and filtered through 0.45-μm filters. In-vitro transduction and determination of the lentivector titre were performer as reported previously58 (link). After 48 h of incubation, the transduced cells were examined microscopically for the presence of TurboGFP expression (70%–90%). To obtain 100% GFP-positive cells, puromycin was added into the medium for an additional 10 days. The reported data are representative of the experiments performed and confirmed using both lentiviral vectors for each gene.