Fig. 4.

CNR2 agonists inhibit nuclear Nrf2 accumulation during hFOB 1.19 cell osteogenic differentiation. hFOB 1.19 cells were cultured at 34 °C until reaching confluence, and then transferred to 39 °C. Forty-eight hours later, HU308 (5, 10, 25, 50 or 100 nM) or JWH133 (1, 2, 5, 10 or 20 μM) was added into the cell medium. Twelve hours later, hFOB 1.19 cells were subjected to a-b western blotting analysis. c For immunofluorescence staining of Nrf2, hFOB 1.19 cells were treated with 50 nM HU308 or 10 μM JWH133 for 12 h. White arrows indicated cells positive to nuclear Nrf2, yellow arrows indicated cells negative to nuclear Nrf2. d-e hFOB 1.19 cells were infected with LV-CNR2 shRNA at 34 °C, and 24 h later, cells were transferred to 39 °C for differentiation. Forty-eight hours later, cells were further treated with 50 nM HU308 or 10 μM JWH133 for 12 h, and then harvested for western blotting analysis