Fig 2: IVA inhibited cardiac fibroblast proliferation and activation. Primary ventricular fibroblasts (PVFs) were pretreated with angiotensin II (Ang II) for 24 h and treated with the indicated dose of IVA for 48 h. (a) The proliferation rate of PVFs in each group was detected by the CCK-8 assay. (b) Protein levels of a-SMA, CTGF, Col 1, Col 3, TGF-ß1, TGFR-2, p-Smad2/3, t-Smad2, and t-Smad3 in the left ventricle of mice detected by western blot analysis. (c) Quantification of a-SMA, CTGF, Col 1, Col 3, TGF-ß1, TGFR-2, p-smad2/3, t-Smad2, and t-Smad3 in (b). Data are presented as mean ± SEM (n = 5 independent experiments); *p < 0.05, one-way ANOVA with the Bonferroni posttest.

Fig 3: Overexpression of FUT8 promoted core fucosylation of TGFBRs and TGF-β1 binding to TGFBRs in MC3T3-E1 cells in the presence of high-dose dexamethasone.MC3T3-E1 cells were transfected with empty or FUT8-overexpressing vectors and cultured in osteogenic medium containing different doses of dexamethasone (0, 10−6, and 10−5 M) as indicated for 3 days. (A) Lens culinaris agglutinin (LCA)-based immunofluorescence staining was performed to detect α1,6-fucosylated trimannose-core structure in MC3T3-E1 cells. Scale bar = 100 µm. (B) LCA blot analysis was performed to determine the level of core fucosylated-proteins. (C) Co-immunoprecipitation was conducted to measure the levels of core fucosylated-TGFBR1 and TGFBR2. (D) MC3T3-E1 cells (from A) were transfected with empty or His-tagged TGF-β1 (His-TGF-β1)-overexpressing vectors. The conditioned medium was collected at 2 days after transfection. MC3T3-E1 cells were cultured in the conditioned medium for 4 h, and the cell lysates were collected. The His-TGF-β1 protein bound to endogenous TGFBRs were detected by the ALP-conjugated anti-His antibody and quantified by measuring the bound ALP using a p-nitrophenyl phosphate substrate. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. empty vector. mOD405, milli-absorbance units at 405 nm. All experiments were independently repeated at least three times.

Fig 4: miR-671 directly targets TGFBR2. a Putative target transcripts of miR-671 predicted using five bioinformatic systems; b A KEGG pathway enrichment analysis based on the above predicted mRNAs; c Expression of miR-671 and the mRNA expression of NF2, FBXW11, DLG, TGFBR2, DVL3, and YWHAZ in mouse cardiomyocytes after miR-671 mimic transfection examined by RT-qPCR; d Putative binding sequence between TGFBR2 and miR-671 and the mutant binding sequence for the construction of luciferase vectors; e Binding relationship between TGFBR2 and miR-671 validated through a dual luciferase assay; f Enrichment of miR-671 and TGFBR2 fragments in the compounds pulled down by anti-Ago2 examined by the RIP assay. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by two-way ANOVA (c, e, and f); *p < 0.05 vs. NC mimic group; ##p < 0.01 vs. anti-IgG.

Fig 5: IVA decreased cardiac fibrosis in HFrEF mice. Nine weeks after TAC surgery, C57BL/6 mice were treated with low-dose (10 mg/kg/d) or high-dose (20 mg/kg/d) IVA for 8 weeks. (a) Representative images of Masson and Sirius red staining of the left ventricle (scale bar, 50 μm) and representative confocal microscopy images of immunofluorescence staining for α-SMA, CTGF, and DAPI (scale bar, 20 μm). (b–d) Quantification of the fibrotic area and quantification of α-SMA and CTGF fluorescence intensity in (a). (e) Protein levels of α-SMA, CTGF, Col 1, Col 3, TGF-β1, TGFR-2, p-Smad2/3, t-Smad2, and t-Smad3 in the left ventricle of mice detected by western blot analysis. (f) Quantification of α-SMA, CTGF, Col 1, Col 3, TGF-β1, TGFR-2, p-smad2/3, t-Smad2, and t-Smad3 in (e). Data are presented as mean ± SEM (n = 9 mice per group); ∗p < 0.05, one-way ANOVA with the Bonferroni posttest.