Fig 2: FSTL1 interacts with canonical Wnt ligands.A, FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 (left panel). Reciprocal IP was performed (right panel). Western blotting was performed on whole lysates and precipitates as indicated. B, FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 (left panel). Reciprocal IP was also performed (right panels). C, FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D, interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F, interaction of FSTL1 and Wnt3a (E) or Wnt10b (F) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 (E) or Wnt10b-V5 (F) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G, ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H, FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I, FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 µg) was incubated with and without recombinant FSTL1 protein (3 µg) in 300 µl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J, domain organization of full-length (FL) and mutant FSTL1 proteins. K, interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L, VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.

Fig 3: Fstl1 +/- mice have less myofibroblast accumulation after silica exposure. (a) Immunofluorescence analysis of a-SMA expression in lung sections of Fstl1 +/- and WT mice 21 days after saline or silica exposure. Representative images of the staining are shown. (a-SMA, green; Endomucin, red; nucleus, blue; scale bars, 200 µm). (b) qRT-PCR analysis of a-SMA mRNA expression in lung tissues from Fstl1 +/- and WT mice 21 days after saline or silica treatment (n = 3 per group; *P < 0.05 by one-way ANOVA followed by Student’s t test). (c) Western blot analysis of a-SMA expression in lung tissues from Fstl1 +/- and WT mice 21 days after saline or silica treatment. ß-tubulin was used as a loading control.

Fig 4: FSTL1 enhances canonical Wnt signaling.A, both mFSTL1 and hFSTL1 proteins augmented mWnt3a mediated 8× TOPflash luciferase reporter activity in HEK293T cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the absence or the presence of mFSTL1 or hFSTL1 (200 ng/ml) before luciferase assay was performed. B, both mFSTL1 and hFSTL1 proteins promoted mWnt3a-mediated 8× TOPflash luciferase reporter activity in NRK-49F cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the presence of increasing amounts of mFSTL1 protein (left panel) or treated with and without mWnt3a protein (10 ng/ml) in the absence or the presence of hFSTL1 protein (right panel), before luciferase assay was performed. C, transfected FSTL1 promoted mWnt3a-induced 8× TOPflash luciferase reporter activity in a dose-dependent manner. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids and increasing doses of FSTL1-HA plasmid. Cells then were treated with or without mWnt3a protein (15 ng/ml) before luciferase assay was performed (right panel). The left panel shows FSTL1 expression in cell culture medium. Albumin was used as loading control. D, transfected FSTL1 potentiated transfected Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, either alone or with FSTL1-HA plasmid, in the absence or the presence of Wnt3a plasmid. About 24 h after transfection, cells were serum starved for 16 h before luciferase assay was performed. E and F, siRNA-mediated inhibition of FSTL1 attenuated Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or two Fstl1 siRNA sequences (siFstl1, 110 nM) either separately (E) or in combination (F). About 24 h after transfection, cells were incubated with or without Wnt3a protein (30 ng/ml) in serum-free medium for 16 h before cells were harvested for luciferase assay. G, the effects of siRNA-mediated inhibition of FSTL1 on Wnt3a signaling were rescued by FSTL1 overexpression. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or Fstl1 siRNAs and with or without FSTL1-HA plasmid. About 24 h after transfection, cells were incubated with or without Wnt3a protein in serum-free medium for 16 h before cells were harvested for luciferase assay. n = 3 to 4. ***p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; hFSTL1, human FSTL1; mFSTL1, mouse FSTL1; NRK-49F, normal rat kidney 49 fibroblast cell.

Fig 5: Fstl1 +/- mice have an attenuated pulmonary inflammatory response. (a–e) Fstl1 +/- and their WT littermate mice were intratracheally exposured to saline or 200 mg/Kg silica, bronchoalveolar lavage fluid (BALF) were collected from Fstl1 +/- and WT mice 7 days after administration of saline or silica. (a) The number of total BALF cells was determined by hemocytometer (n = 7 per group; *P < 0.05 by one-way ANOVA followed by Student’s t test). (b–d) The differential cell counts in BALF were determined according to standard morphologic criteria. (b) Macrophages (n = 7 per group; *P < 0.05 by one-way ANOVA followed by Student’s t test). (c) PMNs (Polymorphonuclear neutrophils, n = 7 per group). (d) Lymphocytes (n = 7 per group). (e) The level of cytokine IL-1ß in BALF was detected by ELISA assay (n = 7 per group; *P < 0.05 by one-way ANOVA followed by Student’s t test). (f–g) Fstl1 +/- and their WT littermate mice were intratracheally exposured to saline or silica, lung tissues were collected from Fstl1 +/- and WT mice 7 days after administration of saline or silica. (f) The level of cytokine IL-1ß in lung tissues was detected by ELISA assay (n = 4 per group; *P < 0.05 by one-way ANOVA followed by Student’s t test). (g) The levels of NLRP3 and caspase-1 (p20) in lung tissues were determined by western blot analysis. ß-tubulin was used as a loading control.