Fig. 7

NPC-derived FGF19 upregulates ANXA2 by blocking TRIM21-mediated ubiquitination. A: ANXA2 expression in HUVECs treated with shFGF19-CM or oeFGF19-CM. B: ANXA2 expression in HUVECs treated with shFGF19-CM or oeFGF19-CM following CHX treatment for the indicated times. C: HUVECs treated with shFGF19-CM or oeFGF19-CM were immunoprecipitated with ANXA2 antibody and analysed by immunoblotting with the anti-ubiquitin antibody to examine ANXA2 ubiquitination. D: Western blot analysis of PI3K/AKT/mTOR in HUVECs treated with shFGF19-CM or oeFGF19-CM. E: Western blot analysis of p-mTOR in HUVECs with the treatment of oeFGF19-CM or the addition of rapamycin. F: HUVECs were transfected with siTRIM21 and treated with shFGF19-CM or NC-CM. Then cells were immunoprecipitated with ANXA2 antibody and analyzed by immunoblotting with the anti-ubiquitin antibody to examine ANXA2 ubiquitination. G: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of HUVECs pretransfected with siTRIM21 and cocultured with shFGF19-CM or NC-CM. H: The relative tube length and number of migrated HUVECs were quantified. I: HUVECs transfected with siTRIM21 or NC and cocultured with shFGF19-CM or NC-CM were mixed with Matrigel for subcutaneous injection. Top: Gross observation of angiogenesis in Matrigel plugs. Bottom: H&E staining was performed to observe blood vessel formation in different groups. J: A working model of FGF19 promoting NPC angiogenesis by influencing TRIM21-mediated ANXA2 ubiquitination through the activation of the PI3K/Akt/mTOR pathway. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001