Hiv 1 packaging plasmids

Lentiviral particles were produced by transfecting HEK293T cells with the third-generation HIV-1 packaging plasmids (Addgene) using Trans IT (Mirus). Cell culture medium containing virus particles was collected at 48 and 72 h after transfection, centrifuged, and filtered through 0.45-µm polyvinylidene fluoride filter (Millipore). Supernatant containing lentivirus was used to infect subconfluent HUVECs. Infected HUVECs were used for experiments at 72 h after infection. All shRNA clones that were used in the study were verified by sequencing (45 clones targeting 22 proteins) and are shown in Fig. S1 B . The identity of 7 clones used in the screen could not be validated by sequencing and is therefore omitted from Fig. S1 B and the phenotype analysis. The efficiency of the knockdown in several cases was assessed by immunoblotting.
For KCTD10 lentiviral overexpression, KCTD10 cDNA was cloned into a CSII-CMV-MCS-IRES2-Bsd vector and packed into lentivirus particles as described previously (Sakaue et al., 2017b (link)). Lentiviral expression and packaging vectors were kindly provided by H. Miyoshi (RIKEN BioResource Center, Wako, Japan). Template cDNA (product ID FHC07641) was purchased from Kazusa DNA Research Institute.

For the rescue experiment, we transduced ECs with shRNA targeting the 3′UTR of FBXW7 (TRCN0000355644; Sigma Mission Library, Sigma-Aldrich). Lentiviral particles were produced by transfecting HEK293T cells with the third generation HIV-1 packaging plasmids (Addgene), using Trans-IT-LT1 (Mirus) as previously described (Kovačević et al., 2018 (link)). For FBXW7 lentiviral overexpression, doxocycline-inducible pTripZ-FBXW7 (generous gift of C. Nicot, University of Kansas Medical Center) was packed into lentiviral particles using the same protocol. To induce the expression of FBXW7, 2 μg/ml doxocycline was added for 24 h to the cell medium.