AT-rich binding protein) [23–26], plays an important role in determination of the location of TTR (replication timing transition region). TTR forms a boundary between adjacent different timing domains, is often present near a synteny boundary, and is associated with higher frequency of SNP and increased occurrence of mutations that are often responsible for cancer [27]. Thus, elucidation of mechanisms for setting of TTR on chromosomes will also provide insight into the generation of mutation hot spots which may ultimately lead to diseases. Materials and Methods Cell Culture Jurkat (Jurkat E6.1; Human T lymphocyte from acute T cell leukemia) and HL-60 (Human promyelocytic leukemia cells) were cultured in RPMI1640 (Nissui) supplemented with 10% fetal bovine serum (FBS), sodium bicarbonate, 3.5 mM L-glutamine, 2- mercaptethanol and antibiotics (100 U/ml Penicillin and 100 mg/ ml Streptomycin (GIBCO)). HeLa S3 (Human epithelial carcino- ma cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, sodium bicarbonate and 5 mM L-glutamine. All the cells were cultured in a humidified atmosphere containing 5% CO2. Jurkat and HL-60 were purchased from ATCC, and HeLa S3 was purchased from the Health Science Research Resources Bank (Tokyo, Japan). Analyses of Replication Timing Replication timing analyses were performed as described previously [20,28,29]. Briefly, exponentially growing cells were pulse-labeled with 50 mM BrdU (5-bromo-2’-deoxyuridine; Roche) for 30 min, fixed in 70% ethanol, stained with propidium iodide (PI) and fractionated by fluorescence assisted cell sorting (FACS Vantage, Becton & Dickinson) into six (G1, S1–S4 and G2) cell cycle fractions, each containing 40,000 cells. After isolation of total genomic DNA, the DNA was sheared by sonication to the average size of 500 bp, denatured and newly replicated, BrdU- labeled DNA was immunoprecipitated using anti-BrdU antibody (BD). To determine the levels of target sequences in each fraction, semi-quantitative PCR was conducted using primers for individual loci and control loci reported previously (Table S1 or primer sequences available on request). The band intensity of the BrdU- labeled target loci was quantified and was normalized by that of the BrdU-labeled mitochondorial DNA (mtDNA) in each cell cycle fraction [30]. PCR products were visualized on 8% acrylamide gels stained with SYBR Green I (Molecular probes). The obtained gel images were captured and band intensities were determined by using LAS-1000 (Fujifilm). Taq polymerase (Sigma- Aldrich or Genescript) or platinum Taq polymerase (Invitrogen) was used for PCR quantification of mtDNA or the genomic target DNA, respectively. FISH Analyses For preparation of nuclei, cells, labeled with 30 mM BrdU for 10 min prior to harvest, were treated with 60 mM (Jurkat) or 75 mM (HL-60 and HeLaS3) potassium chloride (hypotonic condition) for 30–40 min at 37uC and fixed in three changes of ice-cold methanol: acetic acid (3:1). Fixed cells were then dropped onto slides and dried at 55–60uC overnight. To denature chromosomal DNA, slides were preheated at 70uC on a heat block, placed in 70% formamide in 2X SSC at 72uC for 2 min, then transferred quickly to ice-cold 70% ethanol for 5 min, then to 90 and 100% ethanol for 5 min each, and air-dried. BAC clones for the human chromosome 5 were purchased from Invitrogen and were used as probes. The position of each probe is SATB1 Regulates Replication Timing shown in figures. The cosmid clone of cCl12–140 for the human chromosome 11 was kindly provided by Dr. K. Okumura [31]. Biotin-16-dUTP labeled probes were prepared by nick translation. For each slide, 96 ng labeled probe was ethanol-precipitated in the presence of 4 mg human Cot-1 DNA (Roche) and 8 mg salmon sperm DNA (Nacalai tesque) as carrier. DNA was resuspended in 20 ml of hybridization buffer (50% formamide (v/v) and 10% dextran sulfate in 2X SSC), denatured at 80uC for 10 min, and preannealed at 37uC for 20–30 min before applying to the slide. The hybridization probe mixture was applied to each slide under a coverslip, and sealed with rubber solution. Each slide was incubated overnight at 37uC in a moist chamber, and was then washed three times with 50% formamide in 2X SSC for 5 min at 42uC, followed by three washes in 0.8X SSC for 5 min at 60uC. The slides were then incubated for 30 min in a blocking solution (3% bovine serum albumin fraction V (Sigma) and 0.1% Tween20 in 4X SSC) at 37uC and further incubated with anti-BrdU Alexa Fluor 546 conjugate (Invitrogen), Streptoavidin Alexa Fluor 488 conjugate (Invitrogen) and DAPI in a detection solution (1% BSA and 0.1% Tween20 in PBS) for 30 min at 37uC. Then slides were washed three times with PBST (0.1% Tween20 in PBS) for 5 min at 42uC, mounted in an antifade solution (23.3 mg/ml of DABCO (Sigma) and 20 mM Tris-HCl in 90% glycerol) and sealed with nail polish. Slides were stored at 4uC in the dark until observation. To count the numbers of signals, hybridized slides were examined using 63Xoil objective on a Zeiss Axiophot fluorescence microscope fitted with a filter set for DAPI, Alexa Fluor 546 and Alexa Fluor 488. At least 200 S-phase nuclei were scored, and the signal patterns were classified as singlet-singlet (SS)/singlet-doublet (SD)/doublet-doublet (DD) or single (S)/double (D) in some cases. Gene Expression Analyses by RT-PCR Jurkat and HL-60 were stimulated with 20 nM phorbol myristate acetate (PMA) and 1 mM ionomysin for 6 hrs. Total RNA (5 mg) was extracted with Trizol (Invitrogen) according to the manufacturer’s protocol and then reverse transcribed using a oligo(dT)12–18 primer and Expand Reverse Transcriptase (Roche). To prepare non-coding transcripts, total RNA (4 mg) was reverse transcribed using random primers (Takara). PCR amplification of the genes of interest was carried out using the first-strand cDNA. The PCR products were visualized on agarose gel stained with SYBR Green I (Molecular probes). Gel images were captured and band intensities, determined by LAS- 1000 (Fujifilm), were normalized to that of a housekeeping gene, GAPDH. Primer sequences and PCR condition for each primer set are available on request. The list of the genes examined are in Table S3. Genome Analyses through Bioinformatics The genes used in the analysis were taken from NCBI H. sapiens Genome (http://www.ncbi.nlm.nih.gov/). Transposable elements were extracted from the 129.0–132.5 Mb segment of the human chromosome 5 by RepeatMasker analysis (http://www. repeatmasker.org/). Western Blotting and Antibodies Immunoblotting was performed according to standard procedures. Protein samples were mixed with 5X sample buffer (0.25 M Tris-HCl (pH6.8), 10% SDS, 50% glycerol, 11% 2- mercaptoethanol, and 0.02% bromophenol blue), boiled, and separated on SDS-PAGE (7.5 to 10% acrylamide). Antibodies from commercial sources were as follows: SATB1 (ab49061, PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42375