Research Paper Volume 12, Issue 19 pp 19045—19059

Akt activation mediates FGF23-induced osteoblast cytoprotection against DEX. The control OB-6 osteoblastic cells, pre-treated with LY294002 (500 nM, 30 min pretreatment) or the vehicle control (0.1% DMSO), as well as the stable OB-6 cells with the CRISPR/Cas9-Akt1-KO construct (“ko-Akt1”), were treated with FGF23 (25 ng/mL), after 10 min expression of the listed proteins in total cell lysates were shown (A). Alternatively two hours after the FGF23 treatment, cells were treated with dexamethasone (DEX, 1 μM) or the vehicle control (“Veh”), cell viability and cell death were tested by CCK-8 (B) or medium LDH release (C) assays, respectively. The stable OB-6 cells with the constitutively-active Akt1 construct (caAkt1) or the empty vector (“Vec”) were subjected to Western blotting assays to test listed proteins (D). Cells were treated with DEX (1 μM) or the vehicle control (“Veh”), after 48h cell viability (E) and cell death (F) were tested. The primary murine osteoblasts were pretreated with LY294002 (500 nM, 30 min pretreatment), followed by FGF23 (25 ng/mL) treatment for 2h, cells were further stimulated with DEX (1 μM) for 48h, cell viability (G) and death (H) were tested. Data were mean ± standard deviation (SD, n=5). ^# p<0.05. Each experiment was repeated three times and similar results were obtained.