Periostin

The proteins were isolated with RIPA lysis buffer containing protease and phosphatase inhibitors, PMSF, separated by 10–15% SDS-PAGE, and transferred to a PVDF membrane (Millipore, USA). Next, the PVDF membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies against CD9 (1 : 1,000; Cell Signaling Technology), TSG101 (1 : 1,000; Boster), CD81 (1 : 800; Proteintech), calnexin (1 : 1,000; Boster), α-SMA (1 : 1,000; Boster), vimentin (1 : 1,000; Cell Signaling Technology), collagen I (1 : 1,000; Boster), periostin (1 : 1,000; Proteintech), CD31 (1 : 1,000; Arigo), Angptl4 (1 : 800; Cell Signaling Technology), cyclin D1 (1 : 800; Cell Signaling Technology), β-catenin (1 : 800; Cell Signaling Technology), and GAPDH (1 : 2,000; Cell Signaling Technology). The next day, the secondary antibodies (1 : 3,000; Cell Signaling Technology) were incubated with the membranes for 1 h at 37°C. The bands were visualized by enhanced chemiluminescent image analysis.

GQDs were purchased from Nanjing XFNANO Materials Tech Co Ltd (XF152, Nanjing, China). Antibodies to β-actin, Cyclin D1, Cyclin E1, CDK2 MCM5, MCM7, caspase 3, periostin, p-STAT were purchased from Proteintech Group (Wuhan, China). Horseradish peroxidase (HRP)- conjugated anti-mouse and anti-rabbit secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Tublin, DAPI were purchased from ThermoFisher (Shanghai, China). Cell Counting Kit-8(CCK-8) was from Dojindo Laboratories, (Dojindo, Japan). Cell-light EdU Apollo567 In Vitro Kit was purchased from RiboBio (Guangzhou, China). Matrigel Matrix was purchase from BD Biosciences. (Shanghai, China). Isolectin B4 was purchased from Sigma-Aldrich (Shanghai, China). All of the cell culture plates were bought from Corning Life Sciences.

When the density of CFs reached 70–80%, the supernatant was removed and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were blocked with 5% bovine serum albumin (BSA) for 30 min at 37°C and incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1 : 200; Boster, Birmingham, USA), vimentin (1 : 100; Cell Signaling Technology, Danvers, USA), collagen I (1 : 100; Boster), periostin (1 : 100; Proteintech, Rosemont, USA), CD31 (1 : 50; Arigo, Taiwan), and Angptl4 (1 : 100; Cell Signaling Technology) overnight at 4°C. The next day, the fluorescence-conjugated secondary antibodies (1 : 1,000; Cell Signaling Technology) were incubated with cells for 1 h at 37°C. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, USA), and the images were viewed under a fluorescence microscope (OLYMPUS DP73, Tokyo, Japan).