Pgipz lentiviral shrnamir

DAB2IP knockdown cell lines (LAPC4-KD and PC3-KD) were constructed by using the shRNA system (pGIPZ-lentiviral-shRNAmir from Open Biosystems, Huntsville, AL) and selected under puromycin. The cell lines were kindly provided by Dr. Jer-Tsong Hsieh (Department of Urology, University of Texas Southwestern Medical Center) [26 (link)]. LAPC4-KD cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS) (Hyclone, Logan, UT). PC3-KD cells were maintained in RPMI1640 supplemented with 5% FBS. The cells were cultured in a humidified atmosphere containing 5% CO₂ [27 (link)].

Knock-down of SEC23B expression was obtained through infection of lentiviral particles that targeted human SEC23B, in human myeloid leukemia K562 cells, as previously described [5 (link)]. Briefly, we used pGIPZ Lentiviral shRNAmir targeting human SEC23B from Open Biosystems (Horizon Discovery, Waterbeach, UK). We used two different sh-RNAs for SEC23B (V3LHS_357970, sh-70; V3LHS_357974, sh-74) (Table S2). A non-silencing pGIPZ Lentiviral shRNAmir was used as control (RHS4346, sh-CTR). HEK-293T were transfected by 10 µg of sh-RNA plasmid DNA, 30 µL of Trans-Lentiviral Packaging Mix (Open Biosystems Horizon Discovery, Waterbeach, UK) and 25 µL of TransFectin (BioRad, Milan, Italy) in 10-mm plate. The supernatants (10 mL for points) were harvested after 24 h, centrifuged at low speed to remove cell debris and filtered through a 0.45-μm filter. After 48 h of incubation, the transduced cells were examined microscopically for the presence of TurboGFP expression (90–95%). The lentiviral particles of sh-CTR, sh-70 and sh-74 (50 MOI) were used to infect K562 cell line. After 48 h of infection, the cells were maintained in puromycin (0.5 mg/mL) for 2 weeks and then analyzed for GFP+ expression and sorted by cell sorting flow cytometry assay. The sorted GFP+ cells were then assayed for SEC23B expression to verify the stability of the produced clones.

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.