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Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs Sugako Oka, Mizuki Ohno, Daisuke Tsuchimoto, Kunihiko Sakumi, Masato Furuichi, and Yusaku Nakabeppu Supplemental materials Mouse embryonic fibroblasts By means of gene targeting, we previously established Ogg1 gene knockout mice and maintained heterozygous mice (Ogg1+/-) backcrossed to C57BL/6J for 5 generations (Sakumi et al., 2003). MEFs were isolated from embryos (13.5 days postcoital) obtained by mating Ogg1+/- mice, and cultured in DMEM (Invitrogen) supplemented with 10% heatinactivated FBS, 100 µg/ml streptomycin, and 100 units/ml penicillin, at 37˚C in 5% CO2. MEFs were maintained for 40 passages and spontaneously immortalized. Their genotypes were determined by genomic PCR as described previously (Sakumi et al., 2003), and Ogg1+/+ (5L, 9L) and Ogg1-/- (7L, 12L) MEF lines were independently established. Detection of reactive oxygen species In order to confirm that menadione produces reactive oxygen species (ROS) within cells, intracellular ROS such as hydroxyradical or peroxynitrite were detected using hydroxyphenyl fluorescein (HPF, Daiichi Pure Chemicals, Japan). Thirty minutes after exposure to 50 µM menadione, HPF (10 µM) was applied to each MEF culture in fresh medium and the cells were further incubated for 30 min. Grayscale digital images were obtained with an Axio-Skop2-equipped Axio Cam with Axio Vision 3.1 (Carl Zeiss), and are shown in pseudocolor. Construction of plasmids and transfection pcDNA3.1Hyg(-):hOGG1-1a and pcDNA3.1Hyg(-):hOGG1-2a were constructed by inserting the XbaI/BamHI fragment of pET8c:hOGG1-1a and pET8c:hOGG1-2a (Nishioka et al., 1999) into the XbaI/BamHI region of pcDNA3.1 Hyg(-) (Invitrogen). To construct pcDNA3.1PURO:hOGG-1a, a NaeI fragment carrying the coding sequence for hygromycin B phosphotransferase was replaced with a SalI fragment of pPGK-PURU carrying the coding sequence for puromycin-N-acetyltransferase (Tucker et al., 1996). Ogg1-/- MEFs (7L) were transfected with plasmid DNA with the aid of EFFECTENETM Transfection Reagent (Qiagen), according to the manufacturer’s instructions. Stable transfectants were selected in the presence of 250 µg/ml hygromycin B or 25 µg/ml puromycin, and were maintained in the presence of 100 µg/ml hygromycin B or 12.5 µg/ml puromycin, respectively. RT-PCR RT-PCR for Mutyh and Gapdh mRNA was performed as described previously (Ichinoe et al., 2004). Single-stranded DNA index To quantify immunoreactivity of single-stranded DNA, images of immunoreactivity were converted to 256-level gray-scale images, whereas nuclear DAPI staining images were converted to binary monochromes using Adobe PhotoShop 5.0J (Adobe Systems). From the 1 gray-scale images of single-strand DNA immunoreactivity, the signals were detected in areas corresponding to nuclei represented by the monochrome DAPI images, and then measured using NIH Image 1.61. At least 30 cells were analyzed, and an average value of the integrated pixel density per cell was calculated as the index for each sample. Immunostaining Anti-mitochondrial transcription factor A (TFAM) antibody was obtained from Molecular Probes. To verify the specificity of 8-oxoG immunoreactivity in mitochondrial DNA, slides were pretreated with 10 µg/ml MutM protein (Sigma) in nicking buffer (10 mM Tris-HCl, 5 µM ZnCl2, 0.5 mM DTT, 0.5 mM EDTA, 1.5% glycerol, 100 µg/ml bovine serum albumin) or 1000 U/ml of DNase I (Sigma) in reaction buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM MgCl2) at 37˚C for 1 h after RNase treatment. They were then subjected to immunofluorescence microscopy with anti-8-oxoG antibody. Supplemental references Ichinoe A, Behmanesh M, Tominaga Y, Ushijima Y, Hirano S, Sakai Y, Tsuchimoto D, Sakumi K, Wake N and Nakabeppu Y (2004) Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts. Nucleic Acids Res 32: 477-487 Nishioka K, Ohtsubo T, Oda H, Fujiwara T, Kang D, Sugimachi K and Nakabeppu Y (1999) Expression and differential intracellular localization of two major forms of human 8oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs. Mol Biol Cell 10: 1637-1652 Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M and Nakabeppu (2003) Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption. Cancer Res 63: 902-905 Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H, Li E and Jaenisch R (1996) Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev 10: 1008-1020 2