Figure 3
CPUY192018 stimulated the transcription of the Nrf2-ARE-regulated cytoprotective genes in the NCM460 cells in an Nrf2-dependent manner.
(A) Quantitative real-time PCR analysis of Nrf2, HO-1, GCLM and GPx2 in the NCM460 cells. The mRNA levels of Nrf2 and the Nrf2-targeted genes were measured at 10 h after treatment of the NCM460 cells with various concentrations (0.1, 1, 5, 10 μM) of CPUY192018. β-actin was used to normalize the expression of these genes. (B) Western blot analysis of the Nrf2 downstream proteins HO-1, GCLM and GPx2 in the NCM460 cells after treatment with various concentrations (0, 0.1, 1, 10 μM) of CPUY192018 for 8 h. (C) The mRNA expression of Nrf2 and the Nrf2-regulated genes after exposure to Nrf2 siRNA and CPUY192018. The NCM460 cells were treated with Nrf2 siRNA (80 nM), CPUY192018 (10 μM), or Nrf2 siRNA (80 nM) plus CPUY192018 (10 μM). Additional NCM460 cells were treated with a scrambled duplex for use as the blank control. The expression of the Nrf2, HO-1, GCLM and GPx2 genes was quantified using qRT-PCR. (D) Western blot analysis of Nrf2 and the Nrf2-regulated proteins after exposure to Nrf2 siRNA and CPUY192018. The NCM460 cells were treated with Nrf2 siRNA (80 nM), CPUY192018 (10 μM), or Nrf2 siRNA (80 nM) plus CPUY192018 (10 μM). Additional NCM460 cells were treated with DMSO for use as the blank control. The values shown are the means ± SEM (n = 3 independent observations). ***P < 0.001, **P < 0.01 and *P < 0.05, one-way ANOVA with Tukey–Kramer posttest.
