Western Blotting Analysis of Cellular Markers

Western blotting was performed as previously described [18 (link)]. Protein samples were separated by 8–15% SDS-PAGE and transferred to PVDF membranes (C2034, Millipore, USA). After treatment with 5% bovine serum albumin blocking reagent (SW3015, Solarbio, China) for 60 min at 25°C, the membranes were incubated overnight at 4°C with antibodies against LC3 (ab192890, Abcam), P62 (ab109012, Abcam), alpha smooth muscle actin (α-SMA; ab5694, Abcam), and Runx2 (ab76956, Abcam). After being washed thrice with phosphate-buffered saline (Beyotime), the membranes were probed with the corresponding horseradish peroxidase-coupled secondary antibodies (Abcam). Protein bands were visualized using a enhanced chemiluminescence (ECL) detection system (Pierce, USA) and quantified using a gel image analysis system (Bio-Rad, USA).