Anti on

The protein expression was determined by western blot. Monolayer and spheroid HDFCs were resuspended in RIPA lysis buffer (Beyotime) and sonicated. After centrifugation, the protein content was determined in the supernatants by a BCA Kit (Beyotime). A total of 30 μg of proteins from spheroids or monolayer cells was added and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then blotted onto polyvinylidene difluoride membranes. Western blot was performed using anti-Oct4 (1:1000, Cell Signaling Technology, Danvers, MA), anti-Sox2 (1:1000, Cell Signaling Technology), anti-Nanog (1:1000, Cell Signaling Technology), anti-ALP (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-OPN (1:200, Santa Cruz Biotechnology), anti-ON (1:200, Santa Cruz Biotechnology), anti-BSP (1:200, Santa Cruz Biotechnology), anti-DSPP (1:200, Santa Cruz Biotechnology), anti-DMP-1 (1:200, Abcam, Cambridge, MA) and anti-GAPDH (1:2000, Santa Cruz Biotechnology). The membranes were incubated with the primary antibodies overnight at 4°C. After extensive washing, the membranes were further incubated with horseradish peroxidase–conjugated secondary antibodies (1:4000, Abcam) for 1 h. The blots were visualised using an enhanced chemiluminescence BeyoECL star detection kit (Beyotime) and ChemiDoc™ Imaging System quantified band intensities.