Fig 1: FGFR1 knockdown impairs tumor growth and angiogenesis of sorafenib-resistant HCC cells in vivo. (a and b) Pilot studies of tumor xenograft experiments were performed for HUH-7/R cells with 3 groups of treatment: i.e., vehicle control; sorafenib+DJ-1; and sorafenib+DJ-1+shFGFR1. 5 × 106 cells per injection were injected subcutaneously into two flanks of nude mice (n = 3), and tumor weight (a) and tumor volume (b) were recorded. Tumor volume was checked twice to three times weekly using a digital caliper. (c and d) Tumor cell angiogenesis was examined in the chicken chorioallantoic membrane (CAM) model. Both HUH-7 and HUH-7/R cells were mixed with ECDHCC-1 cells at the ratio of 1 : 1 and then treated at different conditions: (1) cells+control; (2) cells+DJ-1; (3) cells+corafenib; (4) cells+sorafenib+DJ-1; and (5) cells+sorafenib+DJ-1+shFGFR1. In CAM model, total vessel length (c) and density (d) were measured at the end of the experiments.
Fig 2: Downregulation of FGFR1 inhibited tube formation and migration of ECDHCC-1 cells stimulated by DJ-1. (a) and (b) Knockdown of FGFR1 by shRNA was confirmed by qRT-PCR and western blotting analysis. (c) Cell viability was analyzed by CCK8. (d–f) The effect of FGFR1 knockdown on DJ-1 inducing angiogenesis on EDCHCC-1 cells was assessed for tube formation assay (d), the number of nodes (e), and total length (f) of capillary tube. n = 3 for all groups. (g) and (h) The effect of FGFR1 knockdown on DJ-1 inducing migration on EDCHCC-1 cells was examined. Photographs (×20) were taken at 12, 24, and 36 h, respectively, after treatment with 15 µg/mL DJ-1 (g). Relative scratch width was quantified by image J software (h). *P < 0.05 and **P < 0.01 versus Con. (original magnifications: ×200 for fluorescence).
Fig 3: Downregulation of FGFR1 affected the apoptosis of sorafenib-resistant HCC cells. (a) Cell viability of THLE-2, HepG2, and HUH-7 after treatment with indicated concentration of sorafenib was assessed by CCK8 assay, and half-maximal inhibitory concentration (IC50) was calculated with a linear fit. (b) Sorafenib-resistant HUH-7 cell line (HUH-7/R) was constructed, and the cell viability was assessed to define the resistance index (RI) of the HUH-7/R cell line. (c) Western blotting analysis of multidrug resistance marker of P-gp and MRP1 was detected. (d and e) Flow cytometry assessment of cell apoptosis by annexin-V/PI (d) and two representative groups after transient knockdown of FGFR1 in HUH-7 and HUH-7/R cells was statistically analyzed (e). (f)–(l) Western blotting analysis of p-FGFR1/FGFR1, cleaved caspase 3, cleaved caspase 9, Bax, and Bcl-2 was carried out in HUH-7 and HUH-7/R cells after transient knockdown of FGFR1 and combined treatment with sorafenib and DJ-1. NS: Not significant, *P < 0.05 versus DJ-1 group, and **P < 0.01 versus the vehicle or DJ-1 alone.
Fig 4: DJ-1 induced FGFR1 phosphorylation and activated the Akt, ERK, and STAT signaling pathways in ECDHCC-1 cells. (a and b) Cells were treated with DJ-1 (15 µg/mL) for 24 h, and then the mRNA expression of FGFR1 (a) and protein expression of phosphorylated FGFR1 (p-FGFR1) and total FGFR1 (b) were detected by qRT-PCR and western blotting analysis, respectively. The inducing activity of DJ-1 was compared with that of EGF (20 ng/mL). (c) Co-immunoprecipitation assay for FGFR1 and DJ-1 was performed. (d–g) Western blotting analysis of Akt, mTOR, ERK, and STAT3 phosphorylation was conducted. The inducing activity of DJ-1 was compared with that of EGF (20 ng/mL). *P < 0.05 and **P < 0.01 versus Con.
Fig 5: DJ-1 induced of angiogenesis and migration in ECDHCC-1 cells. (a) DJ-1 secretion in HCC cells. HepG2 and HUH-7 cells were seeded for culture medium (CM) collection. The CM was harvested and examined by ELISA for DJ-1 expression after 24 and 48 h. (b) Purification of DJ-1 as indicated in the elution lane. (c and d) Product verification of DJ-1 by western blotting analysis with anti-His tag (c) and anti-DJ-1 (d) specific antibody was performed, respectively. (e–g) Fluorescence microscopy imaging of capillary tube formation (e) and quantification of DJ-1 induced tube nodes (f) and tube length (g) using tube formation assay were conducted. (h and i) Wound healing image (h) and quantification of the relative scratch width (i) of ECDHCC-1 cells induced by DJ-1 were analyzed. Con: Control group. *P < 0.05 and **P < 0.01 versus Con. (original magnifications: ×200 for fluorescence).
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