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.
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.
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