Journal: MedComm

Article Title: Generation and utilization of endostatin‐sensitive cell lines for assessing the biological activity of endostatin

doi: 10.1002/mco2.506

Figure Lengend Snippet: Results of potential endostatin‐resistant gene screening by CRISPR/Cas9 gene knockout libraries. ( A ) Western blot–detected expression of large T antigen in the immortalized human umbilical vein endothelial cell (HUVEC) cells using tubulin as the control protein. Antiflag antibody was used to detect large T antigen, large T antigen was constructed on a eukaryotic vector containing flag . ( B ) The HUVEC‐flag‐SV40 proliferation activity at different endostatin concentrations for 2 and 5 days was detected by CCK‐8 (ns, no significant difference; *** p < 0.001). ( C ) Screening process for endostatin resistance genes. sgRNAs were delivered into immortalized HUVEC cells (HUVEC‐flag‐SV40) by lentiviral infection. Under puromycin pressure, the cells were further cultured with/without endostatin for eight generations. RNA sequencing was performed on the zeroth‐ and eighth‐generation cells, respectively. The genes with significantly decreased were selected (UTS2 and NPEPPS), and their expression in HUVEC‐flag‐SV40 cells was downregulated by siRNA, respectively. The red “X” represents UTS2 gene. The blue “X” represents NPEPPS gene. ( D ) The sgRNA enrichment between the dosing group and the control group was analyzed by RNA‐seq, and the corresponding gene loci were assigned. The lower ratio of the dosing group to the control group indicated that the gene deletion increased the cell sensitivity to endostatin.

Article Snippet: Then, plasmid psPAX2, plasmid pMD2G, Human CRISPR Knockout Pooled Library (GeCKO v2), plasmid library A (Addgene, Cat. No. 1000000048), and plasmid library B (Addgene, Cat. No. 1000000049) were cotransfected into HEK293T cells to package and obtain a gene random knockout lentivirus library.

Techniques: CRISPR, Gene Knockout, Western Blot, Expressing, Control, Construct, Plasmid Preparation, Activity Assay, CCK-8 Assay, Infection, Cell Culture, RNA Sequencing