Fig 4.

DJ-1 downregulates DUSP1 expression under an oxidative stress condition. (A) Mouse primary cells were treated with H₂O₂ for 0.25 to 6 h; the expression levels of respective mRNAs were examined by semiquantitative RT-PCR, and their expression levels relative to that of β-actin are shown. (B) HEK293T cells were transfected with FLAG-p53 and DJ-1–HA and treated with 1 mM H₂O₂ for 15 to 45 min (left panel) or 0.5 to 4 h (right panel) at 48 h after transfection. Proteins were analyzed by immunoprecipitation followed by Western blotting. (C) A549 cells were treated with 300 μM H₂O₂ for 15 to 120 min. Proteins were analyzed by immunoprecipitation with an anti-DJ-1 antibody followed by Western blotting. (D and E) DJ-1^+/+ and DJ-1^−/− mouse cells were treated with H₂O₂ for 0.5 h or 2 h. The expression levels of DUSP1 (D) and p21 mRNA (E) were examined by semiquantitative RT-PCR, and their expression levels relative to that of β-actin are shown. (F and G) DJ-1^+/+ and DJ-1^−/− mouse cells were treated with H₂O₂ for 0.5 and 2 h. The expression levels of DUSP1 (F) and p21 mRNA (G) were examined by quantitative RT-PCR. (H) DJ-1^+/+ and DJ-1^−/− mouse cells were treated with H₂O₂ for 0.5 and 2 h. The expression levels of DUSP1, p21, p53, DJ-1, and β-actin were analyzed by Western blotting. (I) DJ-1^+/+ and DJ-1^−/− mouse cells were transfected with control siRNA or p53 siRNA and treated with 300 μM H₂O₂ for 30 min or 2 h at 72 h after transfection. The expression levels of DUSP1 and p21 mRNAs were examined by semiquantitative RT-PCR. The nucleotide sequences of the p53 siRNA are 5′-CCAGAAGAUAUCCUGCCAUTT-3′ (mp53 sense) and 5′-AUGGCAGGAUAUCUUCUGGTT-3′ (mp53 antisense). (J and K) DJ-1^+/+ and DJ-1^−/− mouse cells were treated with H₂O₂ for 0.5 and 2 h. p53 was visualized as described in Materials and Methods. The values shown in panels D through G are means ± SE (n = 3 experiments). **, P < 0.01; ***, P < 0.001. N.S. indicates no significance.