Figure 2.

NMN induces Nrf2-dependent up-regulation of HO-1 and SRXN1 expression in astrocytes. A, B) Confluent cortical astrocytes were treated with 5 mM NMN, and, 24 h later, ChIP was performed with a preimmune IgG (CIgG) or an anti-Nrf2 antibody. Purified DNA was analyzed by real-time PCR with specific primers flanking an ARE sequence in the Hmox1 (A) or Srxn1 promoter (B). *P<0.05, significantly different from vehicle-treated astrocytes. C) Confluent astrocyte cultures were transfected with an NC-siRNA or Nrf2-siRNA. Forty-eight hours later, Nrf2 mRNA levels were determined by real-time PCR and corrected by actin mRNA levels. *P < 0.05, significantly different from NC-treated astrocytes. The lower panel shows decreased Nrf2-protein expression following Nrf2-siRNA treatment in astrocytes. D) Astrocytes were transfected with NC-siRNA or Nrf2-siRNA and, 48 h later, treated with 5 mM NMN. Twenty-four hours after NMN treatment, HO-1 protein levels were analyzed by Western blot. E) Quantification of HO-1 expression after correction by actin levels. F) Astrocytes were treated as in D, and SRXN1 protein levels were analyzed by Western blot. G) Quantification of SRXN1 expression after correction by actin levels. *P < 0.05, significantly different from its respective vehicle (control). For all graph panels, each data bar represents the mean ± sd of at least 3 independent experiments.