Research Paper Volume 12, Issue 19 pp 19045—19059

Nrf2 silencing or KO abolishes FGF23-induced osteoblast cytoprotection against DEX. Genetically-modified stable OB-6 cells with the lentiviral Nrf2 shRNA (“shNrf2) or the lentiCRISPR-GFP-Nrf2 KO construct (“ko-Nrf2”), as well as the parental control cells (“Ctrl”) were treated with FGF23 (25 ng/mL) for applied time periods, expression of listed mRNAs and proteins was shown (A and B); Cells were pretreated for 2h with FGF23 (25 ng/mL), followed by dexamethasone (DEX, 1 μM) stimulation for 48h, cell viability and death were tested by CCK-8 (C) and medium LDH release (D) assays, respectively. Stable OB-6 cells with CRISPR/Cas9-Keap1-KO construct (“ko-Keap1”) were treated with or without FGF23 (25 ng/mL) for 4h, control cells were tranduced with empty vector (“Vec”), expression of listed proteins was shown (E). Alternatively, cells were pretreated for 2h with FGF23 (25 ng/mL), followed by dexamethasone (DEX, 1 μM) stimulation for 48h, cell viability (F) and death (G) were tested. Data are presented as the mean ± standard deviation (n=5). ^# p<0.05. The experiments in this figure were repeated three times, and similar results were obtained.