Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Up-regulation of Fstl1 in silica-injured mice and patients with silicosis. ( a ) Fstl1 mRNA expression in lung tissues of C57BL/6J mice at the different time-point after silica injury was determined by qRT-PCR analysis (n = 6 per group; * P < 0.05; ** P < 0.01 by one-way ANOVA followed by Student’s t test). ( b ) FSTL1 protein in lung tissues of C57BL/6J mice at the different time-point after silica injury was determined by western blot analysis. β-tubulin was used as a loading control. ( c ) Immunohistochemistry (IHC) of FSTL1 in lung sections of C57BL/6J mice 21 days after saline or silica injury. Representative images of the staining are shown. NA stands for normal area, FA stands for fibrotic area; both are shown at higher magnification. (n = 6 per group; scale bars, 200 μm). ( d ) Fstl1 mRNA expression in primary alveolar macrophages (AMs), alveolar epithelial cells (AECs) and fibroblasts (Fb) at day 21 after saline or silica injury was determined by qRT-PCR analysis (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( e ) FSTL1 protein in serum of C57BL/6J mice at the different time-point after silica injury was determined by ELISA analysis (n = 6 per group; * P < 0.05; ** P < 0.01 by one-way ANOVA followed by Student’s t test). ( f ) Representative images of lung fibrotic area of patient with silicosis with H&E staining and immunohistochemical staining for FSTL1 protein (NA stands for normal area, FA stands for fibrotic area; Scale bars, 200 μm). ( g ) FSTL1 levels in serum of patients with silicosis and normal control individuals were determined by ELISA (* P < 0.05 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemistry, Saline, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 +/− mice have an attenuated pulmonary fibrosis phenotype. ( a ) Western blot analysis of FSTL1 expression in lung tissues of Fstl1 +/− and WT mice 21 days after saline or silica injury. β-tubulin was used as a loading control. ( b ) H&E staining of lung sections of Fstl1 +/− and WT mice 21 days after silica injury. Representative images of the staining are shown. Arrows show inflammatory cells. (n = 6 per group; scale bars, 200 μm). ( c ) Lung fibrotic score analysis of the lung sections from Fstl1 +/− and WT mice 21 days after silica injury (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). The fibrotic area is presented as a percentage. ( d ) Hydroxyproline contents in lung tissues from Fstl1 +/− and WT mice were measured 21 days after saline or silica injury (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( e ) Masson trichrome staining of lung sections of Fstl1 +/− and WT mice 21 days after silica treatment. Representative images of the staining are shown (n = 6 per group; scale bars, 200 μm). ( f ) qRT-PCR analysis of Col1a1 mRNA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group; *** P < 0.001 by one-way ANOVA followed by Student’s t test). ( g ) Western blot analysis of type I collagen (Col1) expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group). β-tubulin was used as a loading control. ( h ) qRT-PCR analysis of fibronectin ( Fn1 ) mRNA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Western Blot, Expressing, Saline, Control, Staining, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: 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-1β 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-1β 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. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 +/− mice have less myofibroblast accumulation after silica exposure. ( a ) Immunofluorescence analysis of α-SMA expression in lung sections of Fstl1 +/− and WT mice 21 days after saline or silica exposure. Representative images of the staining are shown. (α-SMA, green; Endomucin, red; nucleus, blue; scale bars, 200 μm). ( b ) qRT-PCR analysis of α-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 α-SMA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica treatment. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Immunofluorescence, Expressing, Saline, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 modulates myofibroblast differentiation via facilitating TGF-β1 signaling. ( a ) qRT-PCR analysis of TGF-β1 mRNA expression in lung tissues of Fstl1 +/− and WT mice at indicated time after saline or silica exposure (n = 4 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( b ) ELISA analysis of active form of TGF-β1 protein in lung tissues of Fstl1 +/− and WT mice at indicated time after saline or silica exposure (n = 4 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( c ) The levels of phosphorylation of Smad2/3 (p-Smad2/3), total Smad2/3 (T-Smad2/3), phosphorylation of Smad1/5 (p-Smad1/5) and total Smad1/5 (T-Smad1/5) in lung tissues of of Fstl1 +/− and WT mice 21 days after saline or silica exposure were determined by western blot analysis. β-tubulin was used as a loading control. ( d ) Primary lung fibroblasts from Fstl1 +/− and WT mice were treated with 5 ng/ml TGF-β1. Protein expressions of α-SMA in cell extracts and type I collagen (Col1) in medium 24 h after TGF-β1 treatment were determined by western blot analysis. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Saline, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: FSTL1-neutralizing antibody attenuates silica-induced lung inflammation and subsequent pulmonary fibrosis in mice. ( a ) In a FSTL1 blockage experiment, C57BL/6 mice were intraperitoneally injected with 22B6 mAb or IgG1 (n = 6 per group) every other day from 1 day after silica challenge till the mice were sacrificed on day 7 for inflammation analysis or day 21 for fibrosis analysis. ( b–f ) For inflammation analysis, ( b ) the number of total BALF cells was determined by hemocytometer (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( c–e ) The differential cell counts in BALF were determined according to standard morphologic criteria. ( c ) Macrophages (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( d ) PMNs. ( e ) Lymphocytes. ( f ) The level of cytokine IL-1β was detected by ELISA assay (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( g – i ) For fibrosis analysis, ( g ) hydroxyproline contents in lung tissues were measured (* P < 0.05, *** P < 0.001 by one-way ANOVA followed by Student’s t test). ( h ) Representative images of the H&E staining of lung sections are shown (Scale bars, 200 μm). ( i ) Lung fibrotic score analysis of the lung sections. The fibrotic area is presented as a percentage (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( j ) Interventional dosing regimen of lung fibrosis model. C57BL/6J mice were intraperitoneally injected with 22B6 mAb or IgG1 (n = 10 per group) at indicated time after silica exposure, and lungs were harvested on day 21. ( k ) Representative images of the H&E staining of lung sections are shown (Scale bars, 200 μm). ( l ) Lung fibrotic score analysis of the lung sections. The fibrotic area is presented as a percentage (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( m ) Hydroxyproline contents in lung tissues (*** P < 0.001 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Clinical characteristics of the study groups.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques:

Journal: Journal of Integrative Agriculture

Article Title: Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

doi: 10.1016/j.jia.2022.07.014

Figure Lengend Snippet: Fig. 3 Lipid metabolism-related pathways were significantly altered upon human follistatin (hFST) overexpression in adipose tissue. A–B, functional enrichment analysis of differentially expressed genes (DEGs) in subcutaneous (A) and retroperitoneal (B) fat. C, heat map of lipid biosynthetic process-related DEGs in two adipose depots. D and E, GSEA of gene sets related to lipid catabolism, lipid synthesis, and lipid storage between transgenic (TG) and wild-type (WT) pigs in subcutaneous (D) and retroperitoneal (E) fat. F and G, hierarchical clustering of the 20 most significant core genes for each gene set between the TG and WT pigs. H, GSEA of eight gene sets between the TG and WT pigs. I and J, heat map of DEGs related to adipose hypertrophy (I) and hyperplasia (J).

Article Snippet: The amount of follistain in serum from the TG and WT pigs were evaluated using a Human Follistatin ELISA Kit according to the manufacturer’s instructions (Elabscience, Wuhan, China).

Techniques: Over Expression, Functional Assay, Transgenic Assay

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 1. A, Plasma follistatin in relation to 3 h of bicycling exercise (n 5). The shaded area marks the 3 h period of exercise. B, Arterial and venous follistatin plasma concentrations during 2 h of one-legged knee-extensor exercise and the following recovery period (n 9). The shaded area marks the 2-h period of exercise. C, Follistatin mRNA expression in skeletal muscle. D, Follistatin mRNA expression in C2C12 muscle cells in response to EPS. E, Follistatin protein release from C2C12 muscle cells in response to EPS. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 2. A, Plasma follistatin in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time point). B, Regulation of follistatin mRNA expression in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time-point). The following tissues were analyzed: spleen, liver, subcutaneous (SubQ) and visceral fat (Vis), soleus, and gastrocnemius (Gastro) muscle, heart, and kidney. C, Western blot on liver tissue from mice performing 1 h of swimming exercise. Time points 1 h, 3 h, and 6 h are depicted as the relative proportion of the 0 h levels. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing, Western Blot

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 4. A, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group). Plasma glucose before and after epinephrine injection. B, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group) Expression of follistatin in the liver in the control and epinephrine- stimulated groups. *, P 0.05. C, Hepatocytes treated with EPS- conditioned media from C2C12 myotubes. CON, Media from non-EPS- stimulated myotubes; MO, negative control consisting of media only; POS, TGF- were used as a positive control. Only TGF- could induce an increase in follistatin mRNA expression.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Clinical Proteomics, Expressing, Control, Negative Control, Positive Control

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 3. A, Infusion of recombinant IL-6 in healthy young men (n 7). A, Plasma IL-6 during a 3-h IL-6-infusion study. The shaded area marks the 3-h period of IL-6 infusion. B, Plasma follistatin during the 3-h IL-6- infusion study. No regulation of plasma follistatin. *, P 0.05. The shaded area marks the 3-h period of IL-6 infusion.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Recombinant, Clinical Proteomics