Research Paper Volume 11, Issue 23 pp 11329—11346

Figure 3. MKP-1 regulates Nrf2 and its target genes in NSCLC xenograft tumors. (A) and (B) Knockdown of MKP-1 reduces the expression of ARE-driven genes in NSCLC xenograft tumors. siMKP1-C1 or siCon cells were injected subcutaneously into Nu/Nu mice and the tumors were removed 6 weeks later. (A) mRNA levels of Nrf2 target genes determined by RT-PCR. The level of 18S rRNA was used as internal control. The value for siCon was set at 100%. Values are mean of three single tumours ± SD, n = 3 (B) Western immunoblots of the expression of MKP-1, Nrf2, and ARE-driven genes with antibodies against the indicated proteins. Each lane represents a single tumor. The relative levels of MKP-1, Nrf2, and ARE-driven genes normalized to actin are shown in right panel. The value for siCon was set at 1. Values are mean of three single tumours ± SD, n = 3. Blots are representative at least three separate experiments. (C) Knockdown of MKP-1 by siRNA against MKP-1 reduces the expression of Nrf2 protein and its target genes in A549 and H460 NSCLC cells. A549 and H460 cells were transfected with each of the MKP-1 siRNAs to Target 1, Target 2, or Target 3. Scrambled siRNA was transfected as negative control. Cells were harvested 48 h later and analyzed by Western immunoblotting with antibodies against the indicated proteins. The relative levels of MKP-1, Nrf2, and ARE-driven genes normalized to actin are shown above each lane. The value for scrambled siRNA-transfected cells was set at 1. Blots are representative at least three separate experiments. **p <0.01 (D) Overexpression MKP-1 increases the expression of Nrf2 protein and ARE-driven genes in A549 cells. (a) A549 cells were transfected with pEGFP-MKP1 or pEGFP vector 24 h before the cells were harvested. Nuclear extracts were probed by immunoblot with anti-MKP-1, anti-Nrf2, or anti-Lamin B1. The relative levels of MKP-1 or Nrf2 normalized to lamin B1 are shown above each lane. The value for pEGFP-transfected cells was set at 1. (b) Luciferase activity in A549 cells transfected with pEGFP, or pEGFP-MKP1. Co-transfections were performed with pGL-GSTA2.41bp-ARE reporter vector and pRL-TK. Dual luciferase activities were analysed. The value for cells transfected with pEGFP plus pGL-GSTA2.41bp-ARE reporter vector and pRL-TK was set at 1. Data are presented as the mean ± SD of triplicate experiments. (E) MKP-1 regulates Nrf2 ubiquitination. A549 cells were transfected with scrambled siRNA or MKP-1 siRNA for 48 h. The cells were exposed to MG132 (20 μM) for 4 h before whole-cell lysates were harvested and subjected to immunoprecipitation with Nrf2. After washing, the immunoprecipitates (Beads) were probed by immunoblotting with anti-UB. The input represents 10% of the total amount of cell lysate use for immunoprecipitation. Results are representative of three separate experiments. *p <0.05, **p < 0.01.