Tgf β1

Tissues from the LV infarct border area (5 mm) and gastrocnemius were homogenized. Total proteins extraction and SDS-PAGE were performed as described before58 (link). The dilution of primary antibodies as follows: FSTL1 (1:1000, GeneTex, Irvine, CA, USA), TGFβ1 (1:500, Bioworld Technology, St. Louis, MN, USA), Smad2/3 (1:1000, Bioworld), p-Smad2/3 (1:500, Bioworld), Akt (1:2000, Cell Signaling, Danvers, MA, USA), p-Akt (1:2000, Cell Signaling), Erk1/2 (1:500, Signalway Antibody, College Park, MD, USA), p-Erk1/2 (1:500, Signalway Antibody), AMPKα1 (1:500, Signalway Antibody), p-AMPKα1 (1:500, Signalway Antibody), Smad1/5/8(1:500, Signalway Antibody), p-Smad1/5/8(1:1000, Cell Signaling) and GAPDH (1:10000, Bioworld). Following incubating with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution, Jackson ImmunoResearch, West Grove, PA, USA), protein bands were subsequently developed with enhanced chemiluminescence. Western quantification was performed by Image Processing and Analysis in Java 1.48 (Wayne Rasband, National Institutes of Health, USA).

The livers and spleens of mice were surgically removed, paraffin-embedded, and sectioned (4 μm). Hematoxylin and eosin (HE) staining was performed to assess the histopathological condition of the tissues and confirm liver metastasis. The localization of IL-10 (Beijing Biosynthesis Biotechnology, Beijing, China) and TGF-β1 (Bioworld Technology Inc., St Louis Park, MN, USA) was visualized by immunohistochemical staining as previously described [22 (link)]. Tissue sections (4 μm) were prepared from formalin-fixed, paraffin-embedded tissues. After deparaffinization and rehydration for antigen retrieval, slides were heated in 10 mM citrate buffer (pH 6.0) in a microwave oven or pressure cooker and cooled to room temperature.

Western blotting was performed as mentioned previously [46 (link)]. Briefly, protein samples were separated in 8–10% SDS-polyacrylamide gel, and then the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (IEVH85R; Millipore, Burlington, MA, USA). The primary antibodies against human or rat DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), mouse DKK1 (AF1096; R&D Systems, Minneapolis, MN, USA), fibronectin (ab45688; Abcam, Cambridge, UK), TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), α-smooth muscle actin (ab7817; Abcam, Cambridge, UK), β-catenin (#4970S; Cell Signaling, Boston, MA, USA), collagen IV (NB120-6586; Novus, Centennial, CO, US) and actin (#4970S; Cell Signaling, Boston, MA, USA) were used in this study. After removing the unbound primary antibody, the membranes were incubated with suitable secondary antibodies. The resultant antigen-antibody complexes were visualized using the Western Lighting Chemiluminescence Reagent (#NEL105001EA; PerkinElmer, Waltham, MA, USA).

Paraffin-embedded rat lung tissue sections were deparaffinized in xylene and rehydrated in graded ethanol washes. Antigen retrieval was performed by cooking tissue sections for 30 min in Tris-EDTA buffer and applying the following primary antibodies: CD68⁺ (ready-to-use, Maixin, Fuzhou, China), CD3⁺ (1:1000, Santa Cruz, California, USA), TGFβ1 (1:800, Bioworld, Louis Park, MN, USA), βCGRP (1:800, ABclonal, Wuhan, China), αCGRP (1:800, ABclonal, Wuhan, China), BAX (1:500, proteintech, Chicago, USA), and PPAR-ɤ (1:800, Bioss, Beijing, China) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies (Dako, Denmark) were used to probe the primary antibodies. Lung fibrosis lesions were classified according to their intensity by Masson’s trichrome into three categories.

For immunohistochemical (IHC) analysis, 5-µm liver sections were deparaffinized, rehydrated, and incubated in 3% H₂O₂ for 20 min to quench endogenous peroxidase activity. After blocking nonspecific staining with normal goat serum, the sections were incubated with polyclonal antibodies against TGF-β1 (1:100, Bioworld, St. Louis, MO, USA) and α-SMA (1:100, Bioworld, St. Louis, MO, USA) overnight at 4 °C. After being washed by PBS, the sections were incubated with goat anti-rabbit antibody at room temperature for 50 min. The antibody binding sites were visualized using 3,30-diaminobenzidine tetrahydrochloride (DAB) and lightly counter-stained with hematoxylin.