Fig 2: Monovalent FSTL3-Fc protein has longer serum half-life and increases muscle mass after systemic administration in mice(A) Schematic presentation of monovalent FSTL3-Fc protein used in this study. See also Supplemental information for cDNA sequence.(B) Ligand neutralization by mono-FSTL3-Fc, measured in Hs578T-reporter cells with 9×CAGA-Luc for SMAD2/3. The data represent mean ± SD from n = 3 independent experiments.(C) Ligand neutralization by mono-FSTL3-Fc, measured in HMEC-1-reporter cells with BRE-Luc for SMAD1/5/8. The data represent mean ± SD from n = 3 independent experiments.(D) Ligand-binding parameters for mono-FSTL3-Fc as determined by reporter cell-based assay. ∗ indicates data from Figure 2C, ∗∗ indicates data from Figure S8A, and ∗∗∗ indicates data from Figure 2D.(E) Immunoblot analysis for FSTL3 in reduced serum taken from mice at indicated time points after i.p. or i.v. injection with mono-FSTL3-Fc (10 mg/kg) (n = 2 independent experiments. #, ψ, Δ, or ▲ represents each mouse).(F) Time course of serum concentration of FSTL3-Fc or control Fc measured by anti-human IgG Fc ELISA. Serum was taken at the indicated time points after injection with monovalent- or bivalent-FSTL3-Fc or control Fc (10 mg/kg). The data represent mean ± SD from n = 3 independent experiments, except for bi-FSTL3-Fc, at 96 hr (n = 2, because of anesthesia-related death). Differences between the conditions were analyzed using analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test for multiple comparison; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig 3: Bivalent FSTL3-Fc is efficiently cleared from mouse circulation(A) Schematic presentation of the protocol. bi-FSTL3-Fc or control Fc was injected into male mice systemically, and blood was temporally collected from the tail at 0‒48 hr after injection (n = 2 independent experiments).(B and C) Immunoblot analysis for FSTL3 in reduced serum taken from mice intravenously (i.v.) (B) or intraperitoneally (i.p.) (C) injected with bi-FSTL3-Fc (10 mg/kg) at the indicated time points (n = 2 independent experiments; the identifying number represents each mouse). See also Figure S1, which contains results of subcutaneous (s.c.) injection (Figure S1B), immunoblot analysis for human IgG Fc of the same sera (Figure S1C), and results of control Fc (10 mg/kg) (Figure S1D).(D) Immunohistochemistry for human IgG Fc in mouse tissues at 5 hr after intravenous injection of bi-FSTL3-Fc. Images are representative of different experiments (n = 2 independent samples), scale bar: 100 μm.

Fig 4: Local administration of bivalent FSTL3-Fc increases muscle mass in healthy mice(A) Schematic presentation of intramuscular injection (i.m.) of bi-FSTL3-Fc into the right calf.(B) Distribution of trypan blue 10 min after i.m. injection into the right calf.(C) Schematic presentation of the protocol. bi-FSTL3-Fc, ActRIIB-Fc, or control Fc was injected intramuscularly into the right calf (hindlimb) of 6-week-old male mice, twice weekly for 2 weeks. All mice were sacrificed 2 days after final injection (n = 10 for each group).(D) Representative macroscopic images of GC muscles excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Muscles on the right hindlimb were injected, while muscles on the left side were used as contralateral counterparts. Images are representative of different experiments (n = 10 independent samples). Scale bar: 1 cm.(E) Normalized weight of muscles excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Muscle weight was normalized to the body weight. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by analysis of variance (ANOVA) with Tukey-Kramer post hoc test for multiple comparison; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. GC, gastrocnemius; QF, quadriceps femoris; TA, tibialis anterior; and Ham, hamstrings; uninjected, left hindlimb; injected, right hindlimb.(F) Representative cross-sectional images of myofibers in the GC muscle excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Top: immunohistochemistry (IHC) for laminin, middle: quantification using Hybrid Cell Count software, bottom: hematoxylin and eosin staining (H&E). Images are representative of different experiments (n = 5 independent samples). Scale bar: 100 μm.(G) Quantification and distribution of muscle fiber cross-sectional area of (F). The differences in muscle fiber cross-sectional area were analyzed using Wilcoxon rank-sum test, and p values were adjusted with Benjamini-Hochberg correction for multiple comparisons; ∗∗∗∗p < 0.0001.(H) Normalized weight of the pancreas of mice after a 2-week treatment. Weight of the pancreas was normalized to the body weight. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by ANOVA with Tukey-Kramer post hoc test; n.s., not significant, ∗∗p < 0.01.(I) The blood glucose concentration of mice after a 2-week-treatment. Mice were fasted for 2.5 hr. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by ANOVA with Tukey-Kramer post hoc test; n.s., not significant, ∗p < 0.05.

Fig 5: Bivalent FSTL3-Fc neutralizes activin A, activin B, GDF8, and GDF11(A and B) Ligand neutralization by bi-FSTL3-Fc, measured in Hs578T reporter cells with 9×CAGA-Luc for SMAD2/3. The data represent mean ± SD from n = 6 (activin A, GDF8, GDF11, and TGF-β3) or n = 3 (activin B) independent experiments.(C) Ligand neutralization by bi-FSTL3-Fc, measured in HepG2 reporter cells with BRE-Luc for SMAD1/5/8. The data represent mean ± SD from n = 3 independent experiments.(D) Ligand neutralization by bi-FSTL3-Fc, measured in HMEC-1-reporter cells with BRE-Luc for SMAD1/5/8. The data represent mean ± SD from n = 3 independent experiments.(E) Ligand-binding parameters for bi-FSTL3-Fc as determined by reporter cell-based assay. (Right) Validation of activation of SMAD signaling pathway after treatment with indicated ligands.