Fig 2: Identification of Sufu as a downstream target gene of miR-378a. (A) The relative luciferase activity of luciferase reports with wild-type or mutant Sufu was determined in ECs, which were transfected with mimic-NC or miR-378a mimic (n = 3). (B) The mRNA expression of Sufu in ECs after transfection with mimic-NC, miR-378a mimic, inhibitor-NC and miR-378a inhibitor was determined by qRT-PCR (n = 3). (C) The protein expression of Sufu in ECs detected using Western blot. (D) Quantitative analysis of the relative protein expression in C (n = 3). *P <.05, **P <.01 vs. the mimic-NC group; # P <.05, ## P <.01 vs. the inhibitor-NC group

Fig 3: P-EVs carrying miR-378a promote the angiogenesis of ECs by downregulating Sufu to activate the Hedgehog/Gli1 signalling pathway

Fig 4: Silencing of Sufu promotes EC proliferation, migration and angiogenesis via Hedgehog/Gli1 signalling activation. (A) The inhibitory efficiency of the siRNAs targeting Sufu was verified by qRT-PCR. (B) The proliferation of ECs treated with PBS, si-NC and si-Sufu #1 was detected by CCK-8 assay (n = 3). (C) The migration of ECs treated with PBS, si-NC and si-Sufu #1 was determined by transwell assay (scale bar, 100 µm). (D) Quantitative analysis of the migrated cells in C (n = 5). (E) The migration of ECs treated with PBS, si-NC and si-Sufu #1 was detected by scratch wound assay (scale bar, 200 µm). (F) Quantitative analysis of the migration rates in E (n = 5). (G) Representative images of the tube formation assay in ECs treated with PBS, si-NC and si-Sufu #1 (scale bar, 200 µm). (H) Quantitative analyses of the total branching points and total tube length in G (n = 3). (I) Detection of the protein level of VEGF in ECs by Western blot. (J) Quantitative analysis of the relative protein expression in I (n = 3). *P <.05, **P <.01 vs. the Control group; # P <.05, ## P <.01 vs. the si-NC group

Fig 5: Knockout of CD56 inhibited Hedgehog and Wnt/β-catenin signaling pathways in 786-O cells. A Pathway enrichment analysis according to CD56 expression in ccRCC by GSEA. B Representative images of the protein of Hedgehog and Wnt/β-catenin signaling pathways measured by western blot. C Grayscale analysis statistics of protein expression levels of Hedgehog signaling pathway. D Grayscale analysis statistics of protein expression levels of Wnt/β-catenin signaling pathway. E Detection of β-catenin expression in cytoplasm and nucleus by immunofluorescence assay. F H&E, CD56, SUFU, β-catenin and c-Myc staining images of subcutaneous tumour tissue in nude mice. All values represented the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (C, D)