t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway

Figure 5

t-BHQ promotes Nrf2 tranlocation from cytoplasm to nucleus and induces the Nrf2-regulated gene expression, especially HO-1. (a) the mRNAs of HO-1, NQO1, GCLC, and Nrf2 were detected by RT-PCR, when cells were treated by t-BHQ (40 μM) for various time (3, 6, 12, or 24 h). (b and c) The immunofluorescent assay was applied to investigate Nrf2 transloction from cytoplasm to nucleus. Scar bar = 20 μm. The content of Nrf2 in nucleus and cytoplasm was analyzed by ImageJ and the ratio of nue/cyt was caculated. in each group. (d) Cells were treated by t-BHQ at various doses (10, 20, 40, and 80 μM) or for various time (3, 6, 12, and 24 h); the protein of HO-1 was detected by Western blot. (e) Cells were pretreated with Act.D (0.5 μg/mL) or CHX (10 μg/mL) for 1 h and then treated with t-BHQ or DMSO for 12 h. Cellular proteins were extracted for HO-1 detection. (f) Cells were transfected with 60 pmols control siRNA or Nrf2 siRNA for 24 h and then treated with t-BHQ (40 μM) for an additional 12 h; the HO-1 protein was detected by immunoblot. Data represent the mean ± SEM of the results. , , and represent significant differences compared with the control group.

(a)

(b)

(c)

(d)

(e)

(f)