Fig 1: Forest plot for the association between FSTL1 levels and calcific aortic valve stenosis in all patients and different subgroups. The association between FSTL1 levels and calcific aortic valve stenosis was analyzed by uni- and multivariable Cox proportional hazard analyses. Log-rank tests were used to compare the impact of FSTL1 on patients. Variables entered in Model 1: age and sex. Variables entered in Model 2: age, sex, hypertension, calcium, Lp (a) and eGFR.
Fig 2: Kaplan-Meier curves for calcific aortic valve stenosis of two groups that were established using the median FSTL1 level. Differences among groups were evaluated with the log-rank test. FSTL1, follistatin-like 1; AS, aortic stenosis.
Fig 3: Effect of different concentrations and durations of FSTL1 treatment on the inhibition of mouse VIC calcification. (A) Different doses (0, 25, 50, and 100 ng/mL) of rhFSTL1 were added to the osteogenic medium for 21 days, and ALP activity and calcium deposition were measured with ALP and Alizarin Red S staining. (B) Quantification of ALP activity was determined by measuring the level of p-nitrophenol in mouse VICs. Data are from 3 independent experiments with 3 technical replicates. **p < 0.01 vs. Con by Student’s t-test. (C) Quantitative analysis of Alizarin red S staining was carried out by incubating stained mouse VICs with cetylpyridinium chloride. Data are from 3 independent experiments with 3 technical replicates. *p < 0.05 vs. Con by Student’s t-test. (D) rhFSTL1 (100 ng/mL) was added to the osteogenic medium for 7, 14, or 21 days. ALP activity and calcium deposition were measured by ALP and Alizarin Red S staining, respectively. (E) Quantification of ALP activity was performed by testing the level of p-nitrophenol in mouse VICs. Data are from 3 independent experiments with 3 technical replicates. **p < 0.01 vs. Con; @p < 0.05, @@p < 0.01 vs. 7 days; and #p < 0.05, ##p < 0.01 vs. 14 days by two-way ANOVA followed by Bonferroni posttest. (F) Quantitative analysis of Alizarin red S staining was determined by incubating stained VICs with cetylpyridinium chloride. Data are from 3 independent experiments with 3 technical replicates. The control group was not treated with rhFSTL1. **p < 0.01 vs. Con; @p < 0.05, @@p < 0.01 vs. 7 days; and #p < 0.05, ##p < 0.01 vs. 14 days by two-way ANOVA followed by Bonferroni posttest.
Fig 4: Effect of FSTL1 on the expression of osteogenic genes in VICs. (A) Different doses (0, 25, 50, and 100 ng/mL) of rhFSTL1 were added to the osteogenic medium for 7 days, and the mRNA levels of Bmp2, Runx2, and Msx2 were measured by RT–PCR. Data are from 3 independent experiments with 3 technical replicates. **p < 0.01 vs. Con by Student’s t-test. (B) Comparison of osteogenic gene expression levels. Data are from 3 independent experiments with 3 technical replicates. *p < 0.05, **p < 0.01 vs. Con by Student’s t-test. (C) Different doses (0, 25, 50, and 100 ng/mL) of rhFSTL1 were added to the calcification induction medium for 7 days, and the protein levels of Bmp2, Runx2, and Msx2 were measured by western blotting. (D) VICs were cultured in the presence or absence of rhFSTL1 (100 ng/mL) and treated with IgG or FSTL1 neutralizing antibody with osteogenic medium for 30 min. Western blotting was used to evaluate the level of phosphorylated ERK1/2. (E) Comparison of ERK1/2 phosphorylation levels. Data are from 3 independent experiments with 3 technical replicates. **p < 0.01 vs. Con by two-way ANOVA followed by Bonferroni posttest. (F) Schematic diagram showing that a reduction in FSTL1 expression leads to phosphorylation of ERK1/2, which then promotes the expression of osteogenic markers and eventually accelerates the progression of calcification. The control group was not treated with rhFSTL1.
Fig 5: Protein levels of FSTL1 in aortic valve samples from non-AS (n = 12) and AS (n = 11) patients. (A) H&E and Masson staining were used to determine the thickness and degree of fibrosis of the aortic valve. Immunofluorescence was used to examine the protein levels and localization of FSTL1 in the aortic valve using vimentin as a marker of VICs. (B) The thickness of the aortic valve leaflet was measured with ImageJ software. **p < 0.01 vs. non-AS by Student’s t-test. (C) Fibrosis of aortic valves was calculated by the percentage of Masson staining percentage tissues. **p < 0.01 vs. non-AS by Student’s t-test. (D) Levels of FSTL1 compared between the two groups. **p < 0.01 vs. non-AS by Student’s t-test. (E) Western blotting was used to evaluate the expression level of FSTL1 in AS (n = 11) and non-AS (n = 12) aortic valve samples. **p < 0.01 vs. non-AS by Student’s t-test.
Supplier Page from Abcam for Human FSTL1 ELISA Kit