Collagen type 3

Dimethyl sulfoxide (DMSO) was acquired from Solarbio Technology (Beijing, China). MEL (purity >95%, molecular weight: 232.28) and protamine sulfate (PS) were purchased from Sangon Bioengineering (Shanghai, China). TGF-β1 was acquired from Ucallm (Wuxi, China). Lipopolysaccharide (LPS) was acquired from Beyotime Biotechnology (ST1470; reagent grade) and purified by phenol extraction (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS), and forward-based medium (FBS) were acquired from XP Biomed (Shanghai, China). HBdSMCs were cultured at 37°C in 5% CO2 and 95% air. The antibodies used included phospho-Smad2 (cat. AF3362, Affinity), phospho-Smad3 (cat. AF3449, Affinity), Smad2/3 (cat. AF6367, Affinity), E-cadherin (cat. 13116, CST), vimentin (cat. 60330, Proteintech), collagen type III (cat. 22734, Proteintech), SQLE (cat. 12544, Proteintech), N-cadherin (cat. 3195, CST), α-smooth muscle actin (cat. 14395, CST), TGF-β1 (cat. 21898, Proteintech), and CCN1 (cat. AF3362, Affinity).

Bladder tissue was fixed in 10% formalin, embedded in paraffin wax and subsequently cut into continuous 5 μm thick sections. To evaluate rat bladder fibrosis, sections were stained using hematoxylin-eosin staining (HE) and Masson’s trichrome staining following standard protocols after dewaxing and washing. The sections were incubated with primary antibody at 4°C for immunohistochemical (IHC) staining, followed by washing, incubation with the secondary antibody coupled with HRP, and a final incubation at room temperature for an additional hour. Phospho-Smad2 (dilution 1:50, cat. AF3362, Affinity), phospho-Smad3 (dilution 1:50, cat. AF3449, Affinity), N-cadherin (dilution 1:50, cat. 13116, CST), vimentin (dilution 1:2500, cat. 60330, Proteintech), collagen type III (dilution 1:500, cat. 22734, Proteintech), E-cadherin (dilution 1:400, cat. 3195, CST), α-smooth muscle actin (dilution 1:1500, cat. 14395, Proteintech), TGF-β1 (dilution 1:200, cat. 21898, Proteintech), and CCN1 (dilution 1:100, cat. AF3362, Affinity) were used.

Concentrations of lysates obtained from apical myocardia (n = 4 hearts per group) were normalized, treated with loading buffer, and proteins denatured at 100°C for 10 min. The proteins were separated were SDS-PAGE gels, transferred on PVDF membranes which were blocked and blotted overnight with the following antibodies: Collagen Type I (Proteintech; 14695-1-AP), Collagen Type III (Proteintech; 13548-1-AP), ANP (Santa Cruz Biotechnology; sc-515701), BNP (Santa Cruz Biotechnology; sc-271185), Cleaved Caspase-3 (Cell Signaling Technology; 9661T) and GAPDH (Proteintech; 10494-1-AP). Western blots were performed in triplicates and normalized with their respective loading controls.