Fig. 3.

Activation of P53 after inhibition of DJ-1. (A) RT-PCR analysis of cultured porcine fibroblast cells after knockdown of DJ-1 by specific siRNA. DJ-1 expression was specifically inhibited by siRNA and, at the same time, activation of MDM2 was observed. Duration (h) after siRNA transfection is indicated. (B) (Left) Activation of P53 after knockdown of DJ-1 in porcine fibroblasts was examined by Western blotting analysis. (Right) Relative band intensities of P53, P53(pS20), and DJ-1 when standardized with Tubulin. P53 and phosphorylated P53 at serine 20 [P53(pS20)] were significantly up-regulated, whereas DJ-1 was successfully down-regulated by knockdown. Error bars, mean ± SEM (t test, n = 3). (C) Transcripts in NT embryos injected with anti–DJ-1 antibody (αDJ1) or IgG (IgG) were examined by RT-PCR analysis. MDM2 was detected only in embryos injected with DJ-1 antibody. (D) Activation of P53 was examined in NT and PA embryos by Western blotting analysis. Anti–DJ-1 antibody was injected into NT embryos (αDJ1, lane 1) and IgG was injected as a control (IgG, lane 2). NT embryos were collected 72 h after nuclear transfer. For Western blots of PA embryos, three types of two-cell stage PA embryos were collected: Nontreated embryos were collected 48 h after parthenogenetic activation (2 cell, lane 3); Mitomycin C was added to the culture medium from 24 to 48 h after activation, and sampling was performed at 48 h (2 cell MC, lane 4); and embryos arrested at the two-cell stage were collected at 120 h (2 cell arrested, lane 5).