Abcom; #61182, BD Biosciences), a-tubulin (T5168, Sigma), peroxide-conjugated anti-rabbit IgG and anti-mouse IgG (711- 036-152 and 711-036-151, respectively, from Jackson Immu- noResearch Laboratories, Inc.). Plasmid Construction Poly2A(+) mRNA of Jurkat was prepared and reverse transcribed as described above. Full-length SATB1 cDNA was amplified by PCR using the following primers; huSATB1-N 59- CCG CTC GAG ATG GAT CAT TTG AAC GAG GCA ACT- 39 and huSATB1-C 59-GCT CTA GAT CAG TCT TTC AAA TCA GTA TTA AT-39. PCR-products, digested with XbaI and XhoI, were gel-eluted and the resulting DNA fragment was inserted at the XbaI-XhoI sites of pCSII-EF-mKO2-Cdt1DXhoI (in which one XhoI to the 59 of mKO2 was deleted; mKO2, monomeric Kusabira Orange 2; [32], generating pCSII-EF-mKO2DXhoI- SATB1. pCSII-EF-mKO2-Cdt1DXhoI digested by XbaI/XhoI was treated with the Klenow fragment and religated, generating a control vector pCSII-EF-mKO2DXhoI. The final clones were verified by sequencing. Transfection and Sorting of Cells Expressing MKO2-SATB1 or MKO2 HeLaS3 cells, plated at a density of 46106 cells per 10-cm plate, were incubated for 24–30 hr prior to transfection. The plasmids encoding mKO2 alone (pCSII-EF-mKO2DXhoI) or mKO2 fused to SATB1 (pCSII-mKO2DXhoI-SATB1) (10 mg each) was in- troduced into HeLaS3 cells using FuGENE HD Transfection Reagent (Roche) according to manufacturer’s protocol. After 24– 48 hrs, cells were observed by Biozero BZ-8000 fluorescence microscope (Keyence, Inc.) to examine transfection efficiency. Then, the cells were labeled with 30 mM BrdU for 10 min prior to harvesting. Cells were collected in PBS containing of 2 mg/ml propidium iodide (SIGMA). Living cells expressing mKO2 were sorted by FACS Aria (BD Biosciences). The sorted mKO2-positive cells were used for replication timing analysis by FISH as described above. Short Hairpin RNA (shRNA)-mediated Knockdown pRS vector or pRS-shSATB1 (ORIGENE) was introduced into Jurkat cells (56106) by electroporation using Nucleofector (AMAXA). The sequences of SATB1-shRNAs are pRS-SATB1- shRNA-1, 59-TCATCAAGTTCTTTCAGAACCAGCGGTAC- 39 and pRS-SATB1-shRNA-2, 59-CAACACA- GAGGTGTCTTCCGAAATCTACC-39. Cells were incubated for 72 hrs and an aliquot of the transfected cells (56105) was used for western analysis to check the repression of expression. The rest of the cells were used for replication timing analyses by FISH as described above. Generation of Stable Cell Lines The pCSII-EF-mKO2DXhoI or pCSII-mKO2DXhoI-SATB1 plasmid was transfected with the packaging plasmid (pCAG- HIVgp) and the VSV-G/Rev-expressing plasmid (pCMV-VSV- G-RSV-Rev) into 293T cells [32] using TransIT293 (Mirus). High-titer viral solutions were collected at 3 days after tansfection, and used for transduction into HeLaS3 cells. At 10 days after transduction, mKO2-positive clones were selected under Biozero BZ-8000 fluorescence microscope (Keyence, Inc.) and HeLaS3 cells expressing mKO2 or mKO2-SATB1 were established and stored. Expression of expected polypeptides was confirmed by Western blot analysis. SATB1 Regulates Replication Timing Chromatin Immunoprecipitation (ChIP) ChIP analysis was carried out in HeLaS3 cells and HeLaS3 expressing SATB1. In brief, cells at about 80% confluency (,16107 cells) were cross-linked for 4 min at 37uC by addition of 1% formaldehyde, and 125 mM glycine was added to terminate the reaction. The cells were washed with ice-cold phosphate- buffered saline (PBS), harvested in buffer I (50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP- 40, 0.2 5% Triton X-100, protease inhibitors and 1 mM phenylmethylsulfonyl fluoride (PMSF)), and incubated at 4uC for 10 min. After centrifugation, pellets were resuspended in lysis buffer II (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA) and incubated at 4uC for 10 min. After centrifugation, pellets were resuspended in lysis buffer III (50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate, and 0.1% SDS containing protease inhibitors and 1 mM PMSF) and sonicated in an ice-water bath until cross-linked chromatin DNA was sheared to an average length around 500 bp. Insoluble materials were removed by centrifugation at 15,000 rpm in a microcentrifuge at 4uC for 5 min. The sonicated cell supernatant was incubated with 10 ml of Protein A Sepharose beads (GE Healthcare) bound with 32 mg of salmon sperm DNA and 2 mg of mouse SATB1 antibody or mouse IgG1 at 4uC overnight. The beads were harvested by centrifugation and washed six times with RIPA wash buffer (50 mM HEPES-KOH (pH 7.0), 500 mM LiCl, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40, protease inhibitors and 1 mM PMSF) and once with TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA). Chromatin antibody complexes were eluted from the beads by addition of elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS). Cross-linking was reversed by incubation at 65uC overnight in TE containing 1%SDS. Protein K digestion was performed for 4 hrs at 37uC in digestion buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 200 mM NaCl, 0.5 mg/ml of proteinase K, and 0.5 mg/ ml of glycogen). ChIP DNA was precipitated with ethanol and dissolved in TE containing RNase A. After incubation for 1 hr at 37uC, ChIP DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. Quantitative Real-time PCR Analysis Real-time PCR was conducted using SYBR Premix EX Taq (Takara) with LightCycler480 (Roche Diagnostics). Primer sequences are available upon request. The PCR products were measured by SYBR green fluorescence. Cell Cycle Synchronization Cell cycle synchronization was conducted as previously de- scribed [33]. Briefly, cells were arrested at the G1/S boundary by incubation in the presence of 2.5 mM thymidine for 16 hrs twice with a 9-hr interval of growth without thymidine. Arrested cells were released into cell cycle and harvested at the indicated times. Preparation and Purification of Genomic DNA 56106 cells were resuspended in 600 ml of lysis buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl and 1 mM EDTA), and 30 ml of 10% SDS and 6 ml of 20 mg/ml Proteinase K was added, followed by gentle mix and incubation at 55uC for several hrs to overnight with rotation. After adding 0.6 ml of phenol/chloro- form/isoamyl alcohol, the samples were mixed and centrifuged at 15,000 rpm for 5 min. The supernatant was treated with an equal volume of chloroform/isoamyl alcohol twice and DNA was precipitated by adding 1/10 volume of 3 M NaOAc and an PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42375