Figure 1. DJ-1-Deficient ES Cells Are Sensitized to Oxidative Stress.

(A) Schematic map of the murine DJ-1 gene in clone F063A04. The retroviral insertion places the engrailed-2 (En2) intron, the splice acceptor (SA), and the β-galactosidase/neomycin resistance gene fusion (β-geo) between exons 6 and 7.

(B) Southern blot analysis of KpnI-digested genomic DNA from DJ-1 homozygous mutant (–/–), WT (+/+), and heterozygous (+/–)cells, probed with murine DJ-1 cDNA. WT DNA shows a predicted 14-kb band (WT), whereas the mutant allele migrates as a 9-kb band (insertion).

(C) Western blot (WB) of ES cell lysates from WT (+/+), DJ-1 heterozygous (+/–), and mutant homozygous (–/–) clones with antibodies to murine DJ-1 (α-DJ-1) or β-actin (α-β-actin). DJ-1 migrates at 20 kDa, β-actin at 45 kDa.

(D) ES cells were exposed to 0, 5, 10, and 20 μM H₂O₂ for 15 h and viability was assayed by MTT. Responses of DJ-1 heterozygous cells (diamonds) and DJ-1 knockout clones 9 (open circles), 16 (solid circles), 23 (squares), and 32 (triangles) are shown. ** p ≤ 0.01; *** p ≤ 0.0001.

(E and F) Cell death of DJ-1 heterozygous and DJ-1-deficient cells (clone 32) after exposure to H₂O₂ (10 μM) was quantified by staining with PI and an antibody to AV with subsequent FACS analysis. AV staining marks cells undergoing apoptosis, whereas PI staining indicates dead cells. * p ≤ 0.05.

(G) DJ-1 heterozygous (+/–) and knockout (clone 32; –/–) cells were assayed at 1, 6, and 24 h after treatment with 10 μM H₂O₂ by Western blotting for cleaved PARP (89 kDa), which indicates apoptosis. No band is seen for cleaved PARP or β-actin for the DJ-1-deficient cells at 24 h due to cell death. Data represent means ± standard error of the mean (SEM) and were analyzed by ANOVA with Fisher's post-hoc test.