Fig 2: FGF15C135P and FGF19P128C are biologically active. A, Reducing and non‐reducing Western blots for FGF19 or Fgf15 of sera from male mice 2 weeks after HDD with 25 μg/mouse of plasmid encoding wild‐type FGF15, FGF15C135P, wild‐type FGF19, or FGF19P128C via HDD. B, Western blot of liver lysate from male mice treated as in A, but sacrificed at 1 week post‐HDD. C, mRNA levels of Cyp7a1 (liver) or Fgf15 (ileum) from male mice at 1 week post‐HDD. Bar graph depicts the relative mRNA levels vs the empty vector control group (assigned a value of 1.0). Mean ± standard deviation, n = 4 for hFc control, and n = 5 for the treatments. PCR reactions were done in triplicate, normalized to beta‐actin. D, IHC with Cyp7a1 antibody from mice treated as in A. 40×.

Fig 3: Divergent Fgf15/FGF19 structures underlie differential ligand activity. A‐D, Western blots of cell lysates from AML12 cells serum‐starved overnight and treated for the indicated times with conditioned medium from Hek293T cells expressing the indicated proteins or empty vector control (1:10 dilution of conditioned media for A and B; 1:5 dilution for C). In panel (D), FGFR1‐3 inhibitor AZD4547 was used at 30 nM and FGFR4 inhibitor BLU9931 was used at 10 nM. E, AML12 cells were serum‐starved overnight then treated for 30 minutes with 1:10 dilution of conditioned medium. Cell lysates were immunoprecipitated with phospho‐tyrosine antibody and FGFR4 was detected by Western blot. Equal loading was determined by Western blot of beta‐actin in pre‐cleared lysate. F, Growth assay of AML12 cells co‐cultured with Hek293T cells expressing the indicated proteins (or empty vector control) for 6 days. Bar graph shows the percent increase in cell number after 6 days (mean ± standard deviation, n = 8). G, Same as F, but with 30 nM AZD4547 and 10 nM BLU9931 as indicated. (*P < .05; ****P < .0001 using ANOVA with Tukey's multiple comparisons test).

Fig 4: FGF19 can synergize with RSPO3 to induce hepatomegaly. A, Liver to body weight ratio from male mice 2 weeks post‐HDD with 50 μg/mouse (25 μg/mouse for FGF single treatment) of plasmid encoding the indicated proteins, fasted for 18h before sacrifice. (*P < .05; **P < .005 using ANOVA with Tukey's multiple comparisons test). B, Mice from A. mRNA levels of Cyp7a1 (liver) or Fgf15 (ileum) from male mice at 1 week post‐HDD. Bar graph depicts the relative mRNA levels vs the empty vector control group (assigned a value of 1.0). Mean ± standard deviation, n = 4 for control and RSPO3; n = 3 for RSPO3 + FGF19, RSPO3 + FGF15; and n = 2 for FGF19 and FGF15. PCR reactions done in triplicate, normalized to beta‐actin. C, Mice from A. Livers sections stained with hematoxylin and eosin (H&E) or antibodies against glutamine synthetase (Glul) or Cyp7a1. Images are centered on the pericentral region. D, β‐hydroxybutyrate and total cholesterol serum levels in mice from A and a matched group fed ad lib. Bar graphs depicts mean +/− standard deviation). n = 4 hFc fasted, RSPO3 ad lib, fasted; n = 3 hFc ad lib, RSPO3 + FGF19 fasted; RSPO3 + Fgf15 ad lib, fasted; and n = 2 RSPO3 + FGF19 ad lib, FGF19 ad lib, fasted, RSPO3 + Fgf15 ad lib, fasted. Each sample measured in duplicate. Data from one of four representative experiments in male and female mice are graphed.

Fig 5: Alterations in structure modulate the ability of Fgf15/FGF1919 to synergize with RSPO3 to induce hepatomegaly. A, Liver to body weight ratios of male mice at 2 weeks post‐HDD with 50 μg/mouse (25 μg/mouse for FGF single treatment) of plasmid encoding the indicated proteins, fasted for 18 hours. hFc n = 13, RSPO3 n = 14, RSPO3 + FGF19 n = 14, RSPO3 + FGF19P128C n = 14, RSPO3 + FGF15 n = 9, RSPO3 + FGF15C135P n = 13, FGF19 n = 6, FGF19P128C n = 6, FGF15 n = 6, and FGF15C135P n = 6. (**P < .005; ****P < .0001 using ANOVA with Tukey's multiple comparisons test. All comparisons in chart.) B, β‐hydroxybutyrate and total cholesterol serum levels in a subset of mice from A. Graphs depict mean ± standard deviation. n = 3 hFc and RSPO3, n = 4 RSPO3 + FGF19P128C and RSPO3 + Fgf15C135P, and n = 5 RSPO3 + FGF19 and RSPO3 + Fgf15. *P < .05, **P < .005; ****P < .0001 using ANOVA with Tukey's multiple comparisons test. C, Liver to body weight ratios of male mice at 2 weeks post‐HDD with 50 μg/mouse (25 μg/mouse for FGF single treatment) of plasmid encoding the indicated proteins, fasted for 18 hours. hFc n = 2, FGF19 n = 4, M70 n = 4, **P < .005 using ANOVA with Tukey's multiple comparisons test.