Enhanced chemical luminescence kit

Cells were lysed in Nonidet P-40 lysis buffer with phenylmethanesulfonyl fluoride and protease inhibitor. Boiled samples of cell lysate with loading buffer were electrophoresed and transferred onto a 0.2μm polyvinylidene fluoride membrane (Millipore, USA). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature followed by incubation with primary antibodies overnight at 4°C. Then the membranes were incubated with secondary antibodies for 1 h at room temperature and were detected with an enhanced chemical luminescence kit (Beyotime technology, China). Antibodies are shown in Supplementary Table 2.

The total protein was extracted using RIPA buffer (Beyotime, Nanjing, China) with protease inhibitor (PMSF, Beyotime, Nanjing, China) on ice for 30 min and collected the supernatant was by centrifuging at 13,450 rpm for 15 min. The total protein concentration was measured using the BCA protein assay kit (Beyotime, Nanjing, China). Total protein extracts (20 µg) from cells were separated via 10% SDS-PAGE (Epizyme, Shanghai, China), transferred to 0.45 mm polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), blocked with 5% non-fat milk for 1 h at room temperature, and washed three times with TBST (5 min each time). The membrane was incubated with the primary antibody overnight at 4 °C, which included rabbit anti-human Collagen I (COL-I, Proteintech, Wuhan, China), and rabbit anti-human RUNX2 (Proteintech, Wuhan, China). On the second day, the primary antibodies were removed and the membrane was cultured with the second antibody for 1 h at room temperature. After incubation with the primary antibody and the secondary antibody, the target protein was visualized by chemiluminescence using an enhanced chemical luminescence kit (Beyotime, Nanjing, China). The ImageJ software was used for the semi-quantification of band intensity.