Fig 2: NPRA positively regulated HIF-1a expression resulting in angiogenesis promotion.A Western blots were employed to assess the HIF-1a expression regulated by NPRA. B Tube formation assays of HUVECs treated with the GC conditioned medium of shCTL, shNPRA, and shNPRA+HIF-1a, respectively, scale bar: 100 µm. C, D Flow cytometry assay and CCK8 assay were used to analyze the HUVEC cell viability and cell proliferation that regulated by the above GC conditioned medium. E qRT-PCR detection showed that NPRA had no significant influences on HIF-1a expression on mRNA level. CM: conditioned medium, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: NPRA promoted angiogenesis by regulating VEGF signaling pathway in GC.A GSEA analysis of the relation of NPRA expression with angiogenesis based on TCGA cohort. B The cluster heat map was used to analyze gene expression levels of subsets of genes in shNPRA GC cells relative to matched shCTL GC cells. C KEGG pathway analysis of the RNA-seq results from shNPRA and the corresponding shCTL GC cells. D GSEA analysis of subsets of genes related to the VEGF signaling pathway.

Fig 4: NPRA promoted GC-associated angiogenesis in vitro.A The expression of VEGF in the culture medium of MKN45 and AGS cells transfected with shNPRA and the control group (shCTL) was detected by ELISA assay. B CCK8 assay was applied to detect cell viability of HUVECs cultured with different conditioned mediums of shNPRA and shCTL GC cells. C Tube-formation assays with HUVECs were performed with the different groups of GC conditioned medium, scale bar: 100 µm. D, E Transwell migration and invasion assays with HUVECs treated with different conditioned mediums showed that knockdown of NPRA reduced the cell ability of migration invasion, scale bar: 200 µm. F, G Flow cytometry assay was used to analyze the HUVEC cell viability and cell cycle regulated by different GC cells conditioned mediums. CM: conditioned medium, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: NPRA promoted tumor angiogenesis by binding and protecting HIF-1a against protein degradation.A The proteins that bound to NPRA were determined by immunoprecipitation/mass spectrum (IP/MS) analysis, which indicated that NPRA could bind to HIF-1a at high binding scores. B Co-IP experiments demonstrated that NPRA immunoprecipitated with anti- HIF-1a antibody, and HIF-1a immunoprecipitated with anti- NPRA antibody. C, D Western blot analysis of HIF-1a protein stability using cycloheximide (CHX, an inhibitor of protein synthesis) when knockdown of NPRA. E AGS cells transfected with shNPRA and shCTL were treated with MG132, HIF-1a, and NPRA protein levels were detected by western blot.