Figure 3.

Celastrol induces Nrf2 activation. (a) RT-PCR analysis of Nrf2 mRNA levels in response to celastrol. Nrf2 mRNA expression peaked after 3 h of celastrol incubation and declined thereafter. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate. (b–g) Nrf2 immunolocalization. Hair cell nuclei were labeled with Hoescht. (b–d) In control utricles Nrf2 is localized to the hair cell cytosol, and the nuclei appear as dark voids (arrows in c). (e–g) Celastrol results in increased Nrf2 immunoreactivity as well as Nrf2 translocation to the nucleus. Scale bar in g=20 μM. (h) qRT-PCR analysis of Nrf2 transcriptional targets. GST, NQO1, UDP, and GGC expression were all increased in celastrol-treated utricles relative to control levels. Error bars represent S.E.M. on real-time qRT-PCRs performed in triplicate