1). mitochondrial ROS as a cytoplasmic complex. or also show radiation hyper-sensitive phenotypes [3]. is mutated in Nijmegen breakage syndrome (NBS), while is mutated in ataxia-telangiectasia-like disorder (ATLD). These genetic disorders show cellular phenotypes similar to AT, suggesting common roles for DDR following IR. The product of has several direct-interaction domains to DDR proteins like ATM and MRE11, thus facilitating DDRs. The product of possesses DNA nuclease activity, which is important for the initial step of HR repair that might lead to chromosome aberrations if defective. Thus, research on these radiation-hypersensitive disorders revealed that the underlying cause of their cellular phenotype was DDR. Although AT, NBS and ATLD share a similar cellular phenotype, some of their clinical manifestations are distinct, particularly the neurodegeneration phenotypes. AT and ATLD patients show progressive cerebellar ataxia, whereas almost all NBS patients show microcephaly [3]. However, the genes involved in cell cycle checkpoints and DSB repair are not accountable for the neurodegeneration phenotypes. AtaxiaCoculomotor apraxia (AOA) is a phenotype related to cerebellar ataxia and is also found in AOA1, AOA2 and AOA3 along with AT and ATLD [4]. Aprataxin, the product of the gene causing AOA1, participates in DNA single-strand break repair [5, 6] while senataxin, the gene product responsible for AOA2, is crucial to resolve DNACRNA hybrid formation (R-loop) in transcript-related DNA damage [7]. In AOA3, is mutated, and the patient-derived cells show abnormal mitochondrial dynamics [8, 9]. Although ATM kinase activation is dependent on DSB damage generation, it can also occur following oxidative stress [3, 10]. Guo and but might be indispensable for the viability of neural cells in the cerebellum. Furthermore, we reported that AOA3-patient cells showed excessive accumulation of ROS (reactive oxygen species), particularly the mitochondria-related ROS superoxide, which perturbed ATM-dependent phosphorylation [8, 9]. We also showed that induction of superoxide by pyocyanin treatment repressed ATM-dependent phosphorylation. Collectively, evidence suggests that the oxidative stress caused by ATM function defects might lead to neurodegeneration phenotypes in AOA3 cells. As ATLD and AT patients show similar neurodegeneration phenotypes, we hypothesized that ATLD cells harbor defects in ATM kinase and that MRE11 may be important for ATM activation upon oxidative stress. We showed here that MRE11 participates in Ziyuglycoside II ATM regulation in response to H2O2- or pyocyanin-induced oxidative stress. We identified FXR1 as a novel cytoplasmic MRE11-binding partner and showed that it also participates in the oxidative stress response. Finally, we discuss the role of MRE11 and FXR1 in cellular response against oxidative stress. MATERIALS AND METHODS Cells and culture HeLa, U2OS, hTERT-immortalized human fibroblasts (48BR), SV40-transformed normal fibroblast (MRC5SV), SV40-transformed ATLD Ziyuglycoside II patient-derived fibroblasts (ATLD2SV and HMfibroSV) and SV40-transformed AT patient-derived fibroblasts (AT5BIVA) Ziyuglycoside II were cultured in Dulbeccos modified Eagles medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics [9, 13, 14]. Mutated in ATLD2SV has a nonsense mutation and expresses C-terminal-truncated MRE11 protein, and mutated MRE11 in HMfibroSV has a missense mutation, in which MRE11 protein is unstable. Normal donor, Tmprss11d AT, ATLD and NBS patient-derived lymphoblastoid cells were cultured in RPMI1640 (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) and antibiotics [9]. Generation of GFP-FXR1-expressing cells Human FXR1 (FBL, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_005087.4″,”term_id”:”1519241653″,”term_text”:”NM_005087.4″NM_005087.4) and MRE11 (MRE11, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_005591″,”term_id”:”1777425444″,”term_text”:”NM_005591″NM_005591) were amplified from a human fetal cDNA library (Clontech) by PCR with Pyrobest DNA polymerase (TAKARA). Then, FXR1 cDNA was inserted into pEGFP-C1 (Clontech) vectors, and MRE11 cDNA was inserted into pCMV-Tag2B-FLAG (Promega) vectors; the insertions were confirmed by DNA sequencing. siRNA knockdown experiments Sub-confluent cells were plated on culture dishes for 24?h and then transfected with siRNAs targeting MRE11 (B-Bridge International Inc.), NBS1 (B-Bridge International Inc.), RAD50 (B-Bridge International Inc.) and FXR1 (Qiagen Co.), or with negative control Ziyuglycoside II siRNA (B-Bridge Ziyuglycoside II International Inc.) using Lipofectamine RNAiMax (Invitrogen Life Technology). Cell were re-plated after 1 day and used for each experiment or immunofluorescence the next day. Antibodies The following antibodies were used in this study: mouse monoclonal anti-H2A histone family X (H2AX) [#05C636], anti-RPA32 [NA-19?L6], anti-RPA70 [NA-18] and rabbit polyclonal anti-H2B [#07C371] (Merck Millipore); rabbit polyclonal anti-FXR1 [A300-892A], anti-phospho-RPA32 [A300-246A], anti-phospho-SMC1 (S966) [A300-050A], anti-SMC1 [A300-055A], anti-RAD50 [A300-184A] and anti-phospho-KAP1 [A300-767A] (Bethyl Laboratories Inc.); rabbit polyclonal anti-phospho-Chk2 (T68) [#2661], anti-phospho-Chk1 (S317) [#2344], anti-phospho-pRad17 (S645) [#6981], anti-phospho-p38MAPK [#4511] and rabbit polyclonal anti-TOMO20 [11802C1-AP] (Proteintech); mouse monoclonal anti-p53-pS15 [#9286].