Fig 2: SerpinE2 can be internalized into cardiac fibroblasts. A. Immunofluorescence assay demonstrated that nystatin and ß-cyclodextrin inhibited the endocytosis of SerpinE2 into cardiac fibroblasts (n = 4, Scale bars = 20 µm). B-C. SerpinE2 was marked by His tag at the C-terminus. Immunofluorescence assay was used to detect the level of His-SerpinE2 and the statistical graph. (n = 9, Scale bars = 10 µm). D-F. mRNA level of collagen I, collagen III and a-SMA. (n = 6); Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (C-F).

Fig 3: SerpinE2 activates ß-catenin signaling pathway in cardiac fibroblasts. A. Co-staining of ß-catenin and serpinE2 in cardiac fibroblasts (n = 4, Scale bars = 20 µm). B. Expression level of ß-catenin was detected by ELISA assay after administration of serpinE2 antibody, heparin, nystatin, ß-cyclodextrin (n = 6); C. mRNA level of ß-catenin in cardiac fibroblasts after administration of serpinE2 antibody, heparin, nystatin, ß-cyclodextrin (n = 6); D. Collagen expression level in CFs after treated with DKK1 (n = 5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (B-D).

Fig 4: The effect of LRP1 and uPAR on endocytosis of serpinE2 in cardiac fibroblasts. A. Protein expression level of collagen I and serpinE2 in cardiac fibroblasts after silencing uPAR (n = 4); B. Protein expression level of collagen I and serpinE2 in cardiac fibroblasts after silencing LRP1 (n = 4); C. Immunofluorescence assay demonstrated that knockdown of LRP1 and uPAR reduced endocytosis of serpinE2 into cardiac fibroblasts (n = 4, Scale bars = 20 µm); D. The level of serpinE2 in supernatant of fibroblast after knockdown of LRP1 and uPAR (n = 8); E. The content of collagen in supernatant of fibroblast after knockdown of LRP1 or uPAR (n = 6); F. It was showed the direct interaction between serpinE2 and uPAR, serpinE2 and LRP1 in cardiac fibroblasts by Co-IP. The panel shows the presence of uPAR and LRP1 in the sample pulled down by anti-serpinE2. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined by one-way ANOVA, Bonferroni's multiple comparisons test (A, B) and Tukey's multiple comparisons test (D, E).

Fig 5: Knockdown of serpinE2 attenuated cardiac fibrosis induced by TAC. A. Masson staining showed the area of fibrosis (Scale bars = 100µm) and transmission electron microscope analysis of heart sections ( Scale bars = 2 µm); B. Fibrotic score was quantified by histological score from Masson staining (n = 4); C. Quantitative analysis of ejection fraction (EF) and fractional shortening (FS) of the heart (n = 5); D. Protein expression level of serpinE2, collagen, p-ERK44/42 and t-ERK44/42 (n = 4); E-G. Relative expression level of serpinE2, collagen and ß-catenin detected by Elisa assay (n = 5-8); Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (B-G).