Fig. 1.

Alterations in CNR2, autophagy molecules, and Nrf2 during osteogenic differentiation in vitro. To stimulate osteogenic differentiation, hFOB 1.19 cells were transferred from 34 °C to 39 °C, and cultured for indicated periods. a ALP activities of hFOB 1.19 cells were determined via a commercial kit. b The expression levels of osteopontin and osteocalcin, two osteogenic markers, were analyzed with real-time PCR. c Cell mineralization was determined with Alizarin red staining. d mRNA expression levels of CNR2 and GAPDH were determined with RT-PCR. e-g Protein levels of CNR2, LC3, beclin 1, p62 and Nrf2 (nuclear and cytoplasmic) were determined with western blotting, and normalized to that of GAPDH or laminB. Symbols*, ** and *** indicated a p value < 0.05, < 0.01 and < 0.001