Anti fluorescence quenching agent

The expressions of α-SMA, p-p65, and p-SMAD in cells were analyzed by immunofluorescence staining. Cells were cultured on glass coverslips at the density of 3,000 cells/cm² and treated with DPP4 with or without GB83 or linagliptin for 48 h. Following treatment for 48 h, cells in the basement layer were washed with PBS and fixed in 4% paraformaldehyde. Following three times washes with PBS for 5 min each, the cells were treated with 0.5% Triton X-100 for 10 min and blocked with bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, and subsequently incubated with α-SMA antibodies (1:100 dilution; Abcam, USA) at 4°C overnight. Cells were washed three times for 5 min each with PBS and then incubated with secondary antibodies (1:200 dilution; Abcam, USA) for another 1 h. Following washing three times for 5 min each with PBS, cells were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature to visualize the nuclei. Following washing with PBS, the slide was mounted with anti-fluorescence quenching agent (Abcam, USA) and cover-slipped; digital images were captured using an inverted fluorescent microscope (magnification, ×400). Digital images were captured four fields per sample with the same exposure time. Three independent experiments were performed.

Cardiac fibroblasts chamber slides were used to detect PRR and YAP protein expression change in different treatments. After all of experiments PRR protein expression and YAP expression were detected by immunofluorescence staining. Cardiac fibroblasts in each group were fixed with 4% paraformaldehyde, blocked with 10% BSA and stained with anti-PRR antibody(diluted ratio 1:250, Abcam, Cambridge, MA) and YAP (diluted ratio 1:200, Cell Signaling Technology, Danves MA, USA) overnight at 4 ℃. PRR protein combined with specific primary antibody was bonded with FITC-conjugated IgG and YAP protein combined with specific primary antibody was bonded with TRITC-conjugated IgG. Cell nucleus were stained with 6-diamidino-2-phenylindole (DEPI) and samples were sealed with anti-fluorescence quenching agent (Abcam, Cambridge, MA). Immunofluorescence intensity was visualized under a Leica fluorescence microscope.