Fig 2: Cardiomyocytes‐derived STC1 inhibits the activities of myocardial fibroblasts in vitro.Cardiomyocytes cells were pretransfected with shSTC1 or shNC, and then the conditioned medium from these transfected cells was used to culture the TGF‐β‐induced myocardial fibroblasts with or without supplementation of re‐STC1. A, Immunofluorescence staining analysis of α‐SMA and COL1A1 expressions in myocardial fibroblasts. Scale bar: 30 or 20 μm. B, Western blot analysis of α‐SMA, COL1A1, and COL3A1 protein expressions in myocardial fibroblasts. Quantification of the gray values of protein bands are shown on the right. n=5. C, Immunofluorescence staining analysis of Smad2 nuclear translocation in myocardial fibroblasts. Scale bar: 20 μm. D, Western blot analysis of Smad2 protein expression in the cytoplasm and nucleus of myocardial fibroblasts. Quantification of the gray values of protein bands are shown on the right. n=5. Data are shown as mean ± SEM. CM indicates conditioned medium; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; re‐STC1, recombinant STC1; shNC, shRNA targeting negative control; shSTC1, shRNA targeting STC1; Smad2, SMAD family member 2; STC‐1, stanniocalcin‐1; TGF‐β, transforming growth factor beta; and α‐SMA, alpha‐smooth muscle Actin.

Fig 3: Cardiomyocytes‐derived STC1 interacts with DOCK1 to reduce the activities of myocardial fibroblasts in vitro. A, STRING database is used to construct a PPI network centered on STC‐1. B, Coimmunoprecipitation assays were performed in cardiac fibroblasts stimulated with re‐STC1 to validate the top candidate binding partners (DOCK1, VEGFA, PAPPA, SLC15A1) identified by the PPI network analysis. C, Coimmunoprecipitation detection confirms that the interaction between STC1 and DOCK1 in myocardial fibroblasts. D, Western blot analysis of DOCK1, RAC1‐GTP, and RAC1 protein expressions in myocardial fibroblasts. Quantification of the gray values of protein bands are shown on the right. n=5. E, Diagram outlining the construction of a series of DOCK1 domain‐deletion mutants. F, Coimmunoprecipitation analysis of the interaction between STC‐1 and different DOCK1 domain‐deletion mutants in myocardial fibroblasts. G, Western blot analysis of the activities of RAC1‐GTP and fibrosis‐related markers (α‐SMA, COL1A1, COL3A1). Quantification of the gray values of protein bands are shown on the right. n=5. Data are shown as mean ± SEM. CM indicates conditioned medium; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; DOCK1, dedicator of cytokinesis 1; IB, immunoblotting; IP, immunoprecipitation; PAPPA, pregnancy‐associated plasma protein A; PPI, protein–protein interaction; RAC1‐GTP: Rac family small GTPase 1 bound to guanosine triphosphate; re‐STC, recombinant STC1; SLC15A1, solute carrier family 15 member 1; STC‐1, stanniocalcin‐1; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; VEGFA, vascular endothelial growth factor A; WT, wild type; and α‐SMA, alpha‐smooth muscle Actin.

Fig 4: STC‐1 interacts with the DOCKER domain inhibits cardiac I/R injury. A, Experimental timeline for evaluating the role of DOCKER domain in STC‐1‐mediated attenuation of I/R‐induced cardiac fibrosis, cardiac fibroblast‐specific AAV9‐TCF21 constructs encoding either wild‐type DOCK1 (AAV9‐TCF21‐DOCK1) or the ΔDOCKER mutant (AAV9‐TCF21‐ΔDOCKER) were administered via myocardial injection 1 week before AAV9‐cTNT‐STC1 treatment in I/R injury. Cardiac functional parameters and fibrotic remodeling were assessed at designated time points. B, Echocardiographic quantification of left ventricular ejection fraction and fractional shortening (n=6/group). C, Representative echocardiographic images. D, Fibrosis assessment by Masson's trichrome staining. Scale bars: 50 μm (200×) and 20 μm (400×). Right panel shows quantitative analysis of fibrotic areas (n=5/group). E, Collagen deposition analysis via Sirius red staining. Scale bars: 50 μm (200×) and 20 μm (400×). Right panel displays fibrotic area quantification (n=5/group). F, Western blot analysis of DOCK1, the activities of RAC1‐GTP, fibrosis‐related markers (α‐SMA, COL1A1, COL3A1), and Smad2 signaling pathway. Quantification of the gray values of protein bands are shown on the right. n=5. Data represent mean ± SEM. AAV9 indicates adeno‐associated virus 9; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; cTNT, cardiac troponin‐T; DOCK1, dedicator of cytokinesis 1; I/R, ischemia–reperfusion; RAC1‐GTP, Rac family small GTPase 1 bound to guanosine triphosphate; Smad2, SMAD family member 2; STC‐1, stanniocalcin‐1; TCF21, transcription factor 21; WT, wild type; α‐SMA, alpha‐smooth muscle Actin; and ΔDOCKER, missing the DOCKER domain.

Fig 5: STC‐1 overexpression mitigates cardiac I/R injury. A, Experimental timeline for evaluating STC‐1's role in I/R injury. Mice received intramyocardial injections of AAV9‐cTNT‐empty vector (control) or AAV9‐cTNT‐STC1 3 wk before I/R surgery. Cardiac functional parameters and fibrotic remodeling were assessed at designated time points. B, Echocardiographic quantification of left ventricular ejection fraction and fractional shortening at postoperative d 25 (n=6/group). C, Representative echocardiographic images corresponding to d 25 measurements. D, Fibrosis assessment by Masson's trichrome staining at d 26. Scale bars: 50 μm (200×) and 20 μm (400×). Right panel shows quantitative analysis of fibrotic areas (n=5/group). E, Collagen deposition analysis via Sirius red staining at d 26. Scale bars: 50 μm (200×) and 20 μm (400×). Right panel displays fibrotic area quantification (n=5/group). Data represent mean ± SEM. AAV9 indicates adeno‐associated virus 9; cTNT, cardiac troponin‐T; EFI/R, ischemia–reperfusion; and STC‐1, stanniocalcin‐1.