Fig. 6: Nrf2 activation is indispensable for Cpd16-mediated osteoblast cytoprotection.
Nrf2 mRNA expression in the listed primary murine osteoblasts was measured (A); the osteoblasts were treated with Cpd16, and listed proteins (B), ARE activity (C), and listed mRNAs (D) were measured; the murine osteoblasts were pretreated with Cpd16 (25 μM) for 2 h, followed by H2O2 (400 μM) stimulation, with cell viability, apoptosis and necrosis tested by CCK-8 (E), TUNEL-nuclei staining (F), and LDH releasing G assays, respectively. Keap1-Nrf2 mRNA expression in the ko-Keap1 or Cas9-C murine osteoblasts was measured (H); the ko-Keap1 murine osteoblasts were also treated with or without Cpd16 (25 μM), expression of listed proteins and mRNAs (I, J) was measured; the ko-Keap1 osteoblasts were pretreated with Cpd16 (25 μM) for 2 h, followed by H2O2 (400 μM) stimulation; cell viability, apoptosis, and necrosis were measured by CCK-8 (K), TUNEL-nuclei (L), and LDH releasing M assays, respectively. *P < 0.05 versus “C” cells. #P < 0.05 versus “shC+Cas9-C”/“Cas9-C” osteoblasts. “N.S.” stands for the non-statistical difference (P > 0.05).
