FGF19 is highly expressed in NPC. A: Representative results of immunohistochemical staining. The first column: IHC detection of FGF19 in nasopharyngeal epithelium tissues. The second and third columns: IHC detection of FGF19 in NPC tissues of stage I-II. The forth and fifth columns: IHC detection of FGF19 in NPC tissues of stage III-IV (top: × 200, bottom: × 400). B: Western blot analysis of FGF19 expression in 3 NPC tissues and 3 nasopharyngeal epithelium tissues. (T) Nasopharyngeal squamous cell carcinoma tissues. (N) Nasopharyngeal epithelium tissues. Tubulin was used as a control for protein load. C: qRT-PCR was used to detect the relative expression of FGF19 in tissues. D: ELISA was used to detect serum FGF19 levels in 61 NPC patients and 36 healthy volunteers. E: Serum FGF19 levels of NPC patients in stage I- II and stage III- IV. F: Serum FGF19 levels in male and female NPC patients. G: Serum FGF19 levels in NPC patients of different ages. Data are presented as the mean ± SD of three independent assessments. *P < 0.05, **P < 0.01, ***P < 0.001, NS: nonsignificant

FGF19 regulates NPC cell malignant behaviours. A: Western blot analysis of FGF19 expression in NPC cell lines (CNE1, CNE2, 5-8F, 6-10B, C666-1) and the immortalized normal nasopharyngeal epithelial cell line NP69. B: ELISA was used to detect FGF19 level in culture medium(CM) of different cells. The column showed FGF19 concentration in CM from NPC cells relative to CM from NP69. C: The interference efficiency of shFGF19 was assessed by western blotting in CNE2 and CNE1 cells. D: CCK8 assay was used to determine cell proliferation after transfection with shNC or shFGF19 in CNE2 and CNE1 cells. E, F: Colony formation assay was performed in shNC or shFGF19 cells. We showed the representative images and the quantification analysis. G, H: Transwell assay was used to determine cell migration in shNC or shFGF19 cells. We showed the representative images and the quantification analysis. I, J: Wound healing assay was performed in shNC or shFGF19 cells. Representative images of cell migration were captured at 0 and 48 h with a microscope. The relative migrated width was calculated by the wound width/the distance measured at 0 h. The histogram showed the relative distance of wound. K: Tg (fli1a: EGFP) transgenic zebrafish were used to evaluate cell metastasis. shNC or shFGF19 CNE2 cells were stained with Dil and injected into the perivitelline cavity of zebrafish at 48 hpf. The migration of tumour cells was evaluated 2 days postinjection. The arrow represented the disseminated foci. We observed the disseminated foci from primary sites under a fluorescence microscope. Data are presented as the mean ± SD of three independent assessments. *P < 0.05, **P < 0.01, ***P < 0.001

FGF19 promotes NPC growth and positively correlates with MVD in vivo. A: CNE2 cells transfected with shNC or shFGF19 were subcutaneously injected into nude mice. Representative pictures of NPC xenografts in nude mice are shown. B: The weights of the excised xenografts in the two groups. C: The volumes of the excised xenografts in the two groups. D: Representative results of immunohistochemical staining of FGF19, CD34 and Ki67 in xenograft sections. Red arrows indicate microvessels. E: Spearman correlation between FGF19 expression and MVD in tumour xenografts. The Pearson correlation coefficient (r²) and P value are shown. F: The column shows relative positive areas of FGF19 and Ki67 in xenografts according to the IHC results. G: Representative results of high and low immunohistochemical staining of FGF19 and CD34 in NPC tissues. Red arrows indicate microvessels. H: Spearman correlation between FGF19 expression and MVD in 10 NPC tissues. Pearson correlation coefficient (r²) and P value are shown. Data are presented as the mean ± SD of three independent assessments. *P < 0.05, **P < 0.01, ***P < 0.001

NPC cells secrete FGF19 into HUVECs and promote angiogenesis. A: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of HUVECs treated with increasing doses of FGF19. B: The relative tube length and number of migrated HUVECs were quantified. C: Morphology of subintestinal vessels (SIVs) in Tg (fli1a: EGFP) transgenic zebrafish after the injection of FGF19 plasmid or negative control. Up: Morphology of subintestinal vessels (SIVs) was photographed by a fluorescence microscope. Bottom: Morphology of SIVs was photographed by a confocal microscope. Arrows: sprouts of SIVs. D: Relative FGF19 level in culture medium(CM) collected from CNE2 cells transfected with shFGF19 or oeFGF19 plasmids. E: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of HUVECs treated with different CMs. F, G: The relative tube length and number of migrated HUVECs were quantified. H: HUVECs pretreated with different CMs 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. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

FGF19 influences ANXA2 expression to accelerate angiogenesis. A: ANXA2 expression in HUVECs with the increasing doses of FGF19. B: The interference efficiency of shANXA2 was assessed by western blotting in HUVECs. C, D: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of HUVECs transfected with shNC or shANXA2. E: Western blotting was used to detect ANXA2 expression with the treatment of FGF19 or shANXA2. F: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of HUVECs. G, H: The relative tube length and migrated HUVECs were quantified. I: HUVECs transfected with shANXA2 or shNC and pretreated with FGF19 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. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

TRIM21 interacts with ANXA2 and triggers ubiquitination. A: ANXA2 expression in HUVECs with or without FGF19 treatment following CHX treatment for the indicated times. B: ANXA2 expression in HUVECs with or without the addition of Mg132 following CHX treatment for the indicated times. C: Co-IP was performed to analyse the interaction between ANXA2 and TRIM21 in HUVECs. IgG was used as a negative control. D: Representative images of colocalization of ANXA2 and TRIM21 in HUVECs. Red: ANXA2; Green: TRIM21. Pearson's R value of scatter plot analysis was calculated using ImageJ. E: ANXA2 and TRIM21 expression in HUVECs after FGF19 treatment was detected by immunofluorescence staining. F: The interference efficiency of siTRIM21 was assessed by western blotting in HUVECs. G: qRT-PCR was used to detect mRNA level of TRIM21 and ANXA2 with the transfection of NC or siTRIM21. H: Western blot was used to detect protein level of TRIM21 and ANXA2 with the transfection of NC or siTRIM21. I: ANXA2 expression in HUVECs transfected with NC or siTRIM21 following CHX treatment for the indicated times. J: HUVECs transfected with NC or siTRIM21 were immunoprecipitated with ANXA2 antibody and analysed by immunoblotting with the anti-ubiquitin antibody to examine ANXA2 ubiquitination. Whole-cell lysates were used for western blotting with an anti-TRIM21 or anti-ANXA2 antibody. K: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of shANXA2-HUVECs transfected with NC or siTRIM21. L, M: The relative tube length and number of migrated HUVECs were quantified. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

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