Fig. 2: FBXO22 promotes angiogenesis in liver cancer both in vitro and in vivo.

A Tube formation assays were conducted by culturing HUVECs on Matrigel with tumor-conditioned medium (CM), and the tube-forming ability was evaluated by quantifying the number of junctions formed 4–6 h after treatment with CM. B Representative images and the average number of HUVECs junctions cultured by Hep3B FBXO22 overexpression cell line and MHCC97H FBXO22 knockdown cell line. C Representative images and number of migration or invasion HUVECs cells cultured by CM derived from Hep3B FBXO22 overexpression cell line and MHCC97H FBXO22 knockdown cell line. D, E Fluorescence images and a liver diagram (left panel) depicting representative data, along with quantitative results of fluorescence intensity and tumor volume (right panel), were obtained from the Mock, FBXO22OE, shControl and shFBXO22 groups in an intrahepatic model. F Overexpression of FBXO22 resulted in increased gene expression of well-known pro-angiogenic factors, with the most notable upregulation observed in VEGF-A. Total RNA was extracted from Hep3B cells 48 h after plasmid transfection and analyzed using qRT-PCR. G Western blotting showed that downregulation of FBXO22 in MHCC97H cells resulted in decreased HIF-1α and VEGF-A levels. In contrast, upregulation of FBXO22 in Hep3B cells resulted in increased HIF-1α and VEGF-A expression. H Quantitative results of immunohistochemical staining of FBXO22 and angiogenesis-related genes (CD31 and VEGF-A) in intrahepatic tumor models. I The expression of FBXO22 in tumor cells of liver cancer tissue is positively correlated with VEGF-A levels. To assess this relationship, consecutive sections of HCC tissue were embedded in paraffin and subjected to IHC staining for the detection of FBXO22 and VEGF-A expression, along with standard image analysis techniques for patient IHC staining. J The addition of VEGF-A neutralizing antibodies to tumor-conditioned medium inhibited tubule formation in HUVECs induced by overexpression of FBXO22. Conditioned media from Hep3B and HepG2 cells overexpressing FBXO22 were collected and supplemented with 3.2 μg/ml of anti-VEGF-A to induce tubule formation in HUVECs, followed by statistical analysis. Data and error bars are presented as mean ± SD from triplicate independent replicate experiments. The data were analyzed using a paired Student’s t-test for experiments (B, C, D, E, F, H and J). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.