Fig 2: The mRNA and protein expression levels of FGF19/FGFR4 after FGF19 knocking down. The mRNA (A,B) and protein expression levels of FGF19/FGFR4 (C,D) after hFGF19 knocking down. The invasion and migration ability of cell was determined by trasnwell migration assays at 24 h (F), transwell invasion assays at 36 h (F) and wound healing at 12 h, 24 h, 36 h (E) (Scale bars: (E,G), 100 nm). Quantitative data from three independent experiments are shown in the bottom, respectively (G,H). * p < 0.05; ** p < 0.01, *** p < 0.001.

Fig 3: FGF signaling promotes lysosome biogenesis and ER-phagy in chondrocytes through FGFR3 and FGFR4 AMS proteomic analysis of RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/ml) for 16 h. Biological processes and cellular components regulated by FGF signaling are shown. FDR < 0.05. N = 4 biological replicates/treatment were analyzed. P-value was calculated using 1D annotation enrichment test based on Wilcoxon–Mann–Whitney test.B, CFACS analysis of LysoTracker (B) and DQ-BSA (C) dye fluorescence in RCS treated with the indicated FGF ligands (50 ng/ml) for 16 h. Concanamycin A was used at 100 nM for 1 h to inhibit lysosomal function. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment. One-way analysis of variance (ANOVA) P < 0.002 (B) and P = 0.005 (C); Tukey's post hoc test ***P < 0.0005.DLamp1 immunofluorescence (red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/ml) for 16 h. Insets show magnification of the boxed area. Nuclei were stained with DAPI (blue). Scale bar 15 and 2 µm (higher magnification boxes). Bar graph shows quantification of Lamp1-positive vesicles/cell. Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment. n = 45 cells were analyzed. Student's paired t-test *P < 0.05.ERepresentative TEM images of RCS chondrocytes treated with 5% ABS (vehicle) and FGF18 (50 ng/ml) for 16 h. BafA1 (100 nM; 4 h) was used to inhibit lysosome activity. Arrows indicate lysosomes. Higher magnification insets showed the presence of ER membranes decorated with ribosomes (arrowheads). Scale bar 500 nm. Quantification shows average number of ER membranes and mitochondria number/lysosome vesicle (Lys). Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment. ER fragment/vesicle n = 60 (vehicle) and n = 72 (FGF18) cells were analyzed; mitochondria number/vesicle: n = 40 (vehicle) and n = 72 (FGF18) cells were analyzed. Student's paired t-test ***P < 0.0005; NS, not significant.FRepresentative co-immunofluorescence staining of ER (ER-Tracker BODIPY Green) and lysosomes (Lamp1, red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/ml) for 16 h. Insets show higher magnification of boxed area. Scale bar 15 and 2 µm (higher magnification boxes). N = 3 biological replicates/treatment.GSchematic representation of EATR assay: eGFP fluorescence, but not mCherry, is lost at acidic pH. Chondrocytes with indicated genotypes (ctrl = wild type) were treated with FGF18 (50 ng/ml; 12 h) and BafA1 (200 nM; 3 h) where indicated. ER acidification was measured by FACS. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. One-way analysis of variance (ANOVA) P < 0.0001; Tukey's post hoc test ***P < 0.0005; **P < 0.005; NS, not significant.HFACS analysis of DQ-BSA dye fluorescence in chondrocytes with indicated genotypes (ctrl = wild type) treated with FGF18 (50 ng/ml) for 16 h. Concanamycin A was used at 100 nM for 1 h to inhibit lysosomal degradation. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment/genotype. Analysis of variance (ANOVA) P = 0.00279; Tukey's post hoc test **P < 0.005; *P < 0.05; NS, not significant.IEATR assay. Chondrocytes with indicated genotypes (ctrl = wild type) treated with FGF18 (50 ng/ml; 16 h) where indicated. ER acidification was measured by FACS. Mean ± standard error of the mean (SEM) of N = 17 (veh), N = 17 (FGF18), N = 4 (FGFR3;4KO); N = 4 (FGFR3;4KO FGF18) biological replicates. One-way analysis of variance (ANOVA) P < 0.0001; Tukey's post hoc test ***P < 0.0005; NS, not significant.JRepresentative immunofluorescence staining of ER (CLIMP63, green) and lysosomes (TMEM192-HA, red) in FGFR3;4KO chondrocytes treated with vehicle (5% ABS) and FGF18 ( 50 ng/ml for 16 h). BafA1 was used at 100 nM for 4 h to inhibit lysosomal degradation. Quantification of relative CLIMP63 fluorescence into TMEM192-HA-positive vesicles. N = 3 biological replicates; n = 21 cells were analyzed. Mean ± standard error of the mean (SEM). Paired Student's t-test NS = not significant. Scale bar 10 and 1.3 µm (higher magnification boxes).

Fig 4: The effect of melatonin on cell migration and invasion in SCC-15 cells with or without FGF19 overexpression. The mRNA (A,B) and protein expression levels of FGF19/FGFR4 (C) after hFGF19 overexpression. The invasion and migration ability of cell was determined by trasnwell migration assays at 24 h (G), transwell invasion assays at 36 h (G) and wound healing at 12 h, 24 h, 36 h (E) (Scale bars: (E,G), 100 nm). Quantitative data from three independent experiments are shown in the right panels, respectively (D,F,H). * p < 0.05; ** p < 0.01, *** p < 0.001.

Fig 5: Rationale for cell-based receptor complementation assay and structure-based sequence alignment of seven FGFR isoforms.a-b, Rationale for receptor complementation assay. Center: Cartoon/surface representation of the FGF23–FGFR1c–αKlotho–HS quaternary complex in two orientations related by 180° rotation around Y axis. Circles denote approximate circumferences of ligand binding sites in primary and secondary receptors selected for mutagenesis. a, Left: expanded view of circled region on the secondary receptor showing that Ile-203 and Ser-223 of FGFR1cS engage in hydrophobic and hydrogen bonding contacts with residues in FGF23’s core. Right: expanded view of circled region on the primary receptor showing that Ala-167 and Val-248 of FGFR1cP engage in highly conserved hydrophobic contacts with Tyr-124 and Leu-158 of FGF23, respectively. Additionally, Ala-167 also makes a hydrogen bond with Tyr-124. b, Left, expanded view of the circled region on the primary receptor showing that the corresponding Ile-203 and Ser-223 in FGFR1cP are solvent exposed. Thus, an engineered FGFR1cI203E/S223E molecule (i.e., FGFR1cΔSLBS) should preserve the ability to act as primary receptor despite losing the capacity to function as secondary receptor. Right, expanded view of the circled region on secondary receptor showing that the corresponding Ala-167 and Val-248 in FGFR1cS do not play any role in the quaternary complex formation and are solvent exposed. Thus, an engineered FGFR1cA167D/V248D mutant (i.e., FGFR1cΔPLBS) is predicted to lose the ability to function as primary receptor but could still act as secondary receptor. c, Demonstration of receptor complementation between FGFR1cΔSLBS and FGFR1cΔPLBS and between FGFR4ΔSLBS and FGFR1cΔPLBS via PLA. Representative fluorescent microscopy fields of stable cell lines subjected to PLA are shown. Scale bar,10 μm. Experiments were performed at least two times biological repeat with similar results. d, Structure-based sequence alignment of ligand binding regions (i.e., D2, D2-D3 linker and D3) of all seven FGFR isoforms. As a guide, a schematic representation of a prototypical FGFR is shown and its various domains are labeled. The C-terminal half of D3 which undergoes alternative splicing in FGFR1-FGFR3 is highlighted in dark blue. Note that the unspliced N-terminal half of D3 is conserved between b and c isoforms. Secondary structure elements are provided on the top of alignment. The HS interacting residues are all localized within D2 domain and are colored in magenta. Residues mediating the FGF23-FGFRP and αKlotho-FGFRP interfaces are colored yellow and green, respectively. Note that residues mediating direct FGFRP-FGFRS contacts (in light blue) are fully conserved amongst all seven isoforms. As for the FGF23-FGFRS interface (in orange), only D2 residues that contact FGF23’s core, are conserved between the seven isoforms. However, two hydrophobic D3 residues that interact with FGF23 N-terminus at the FGF23-FGFRS interface are conserved only in FGFR1c-3c and FGFR4 (indicated by black arrow heads). In FGFR1b-3b, these residues, which overlap with binding site for RBA of αKlotho, are replaced by charged residues. Hydrophobicity of D3 groove FGFR1b-3b is further disrupted by the presence of a unique N-linked glycosylation site within their D3 grooves (denoted by a red box). In FGFR2c, a key conserved hydrophobic residue is replaced by a charged Lys-296 (underlined in red) which likely accounts for the inability of FGFR2c to bind αKlotho.