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. 2021 May 3;41(6):511–527. doi: 10.1002/cac2.12157

FIGURE 3.

FIGURE 3

PD‐L1 combines with VEGFR2 directly and regulates angiogenesis. (A) GSEA analysis was performed using PD‐L1‐knockdown and control cells (OVCA433/PD‐L1‐sh1 and OVCA433/control). A gene signature was defined as a gene with significant expression changes. (B) and (C) Effect of PD‐L1 silencing on tube formation of HUVECs. HUVECs were treated with cell supernatant gained from OVCA433/PD‐L1‐sh1, OVCA433/PD‐L1‐sh2, and control cells. (D) ELISA of VEGFA in PD‐L1‐knockdown ovarian cancer cells and control cells. (E) Cellular extracts from OVCA433 cells were immunopurified with anti‐PD‐L1 affinity columns and eluted with PD‐L1 peptide. The eluates were resolved by SDS‐PAGE and silver stained. IgG was the negative control. (F) Co‐IP analysis demonstrates an interaction between PD‐L1 and VEGFR2 in ovarian cancer cell lines. (G) Immunofluorescence assay demonstrates the relationship between PD‐L1 and VEGFR2. (H) Western blotting analysis of proteins associated with cancer metastasis and angiogenesis in PD‐L1‐silenced ovarian cancer cells and control cells. β‐actin was used as a loading control. (I) Heat map of qRT‐PCR analysis of indicated genes in PD‐L1‐knockdown and control OVCA433 cells. **P < 0.01. Abbreviations: PD‐L1: programmed cell death‐ligand 1; VEGFR2: vascular endothelial growth factor receptor 2; GSEA: gene set enrichment analysis; PD‐L1‐sh1/2: PD‐L1 short hairpin RNA 1/2; ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate; HUVEC: human umbilical vein endothelial cell; ELISA: enzyme‐linked immunosorbent assay; VEGFA: vascular endothelial growth factor A; OD: optical density; SDS‐PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; Co‐IP: co‐immunoprecipitation; DAPI: 4′,6‐diamidino‐2‐phenylindole; IB: immunoblotting; qRT‐PCR: quantitative real‐time polymerase chain reaction; FAK: focal adhesion kinase; AKT1: protein kinase B; SCL: stem cell leukemia; Tie1: tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1; Smad1: mothers against decapentaplegic homolog 1; C‐MET: cellular‐mesenchymal epithelial transition factor; PDGFA: platelet‐derived growth factor A; Ang1: angiopoietin 1; Ang2: angiopoietin 2