PMC:1072800 / 6192-13581 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/1072800","sourcedb":"PMC","sourceid":"1072800","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1072800","text":"MATERIALS AND METHODS\nSynchronization of HaCat cells, mRNA isolation and northern blot hybridization were carried out as described previously (48).\n\nSite-directed mutagenesis of hOGG1\nFull-length hOGG1 cDNA cloned into pEGFP-N1 vector (pHOGH1-EGFP-12) as described previously (14), represents the wild-type hOGG1 construct, referred to as pEGFP-hOGG1. The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce the Ser326Cys (pEGFP-hOGG1-Cys326), the Ser326Ala (pEGFP-hOGG1-Ala326) and the Ser326Glu (pEGFP-hOGG1-Glu326) mutations with pHOGH1-EGFP-12 construct as the parental vector according to the manufacturer's protocol. The cloned fragments were sequenced and the desired mutations were thus verified. The pEYFP-hOGG1 plasmid was constructed by ligating the hOGG1 cDNA fragment from pEGFP-hOGG1 into the pEYFP-N1 vector. The pECFP-PCNA and the HcRed-PCNA are described in (49) and (50), respectively.\n\nCell culture, DNA transfection and selection of stable cell lines\nHeLa S3 cells were cultivated in DMEM with 4.5 g/l glucose, 10% FCS, 0.3 mg/ml glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in a 5% CO2 air atmosphere. Transfections of HeLa S3 cells were performed using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals) according to the manufacturer's recommendations. To generate stable cell lines, 8 × 105 HeLa S3 cells were plated in 100 mm culture dishes. After overnight incubation, cells were transfected with pEGFP-hOGG1, pEGFP-hOGG1-Cys326, pEGFP-hOGG1-Glu326 and pEGFP-N1. After 2 days, each plate was subcultured into six 100 mm culture dishes. Cells were grown overnight and 800 μg/ml geneticin was added. Single green fluorescent clones were isolated after 20 days.\n\nNuclear protein extracts, western blot and assay for faPy DNA glycosylase activity\nNuclear extracts from HeLa-EGFP, HeLa-EGFP-hOGG1, HeLa-EGFP-hOGG1-Cys326 and pEGFP-hOGG1-Glu326 were prepared as described previously (28). For western blot analysis, 20 μg nuclear protein extracts were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. To detect EGFP and EGFP-hOGG1 (and the mutants) the membrane was probed with a polyclonal anti-GFP antibody (ab290-50; Abcam), and to detect EGFP-hOGG1 (and the mutants) a monoclonal anti-human OGG1 antibody (clone 7E2; IBL) was used. FaPy DNA glycosylase assay using increasing amounts of nuclear extracts was carried out as described previously (48).\n\nCell-cycle synchronization, flow cytometry and immunofluorescence\nHeLa-EGFP-hOGG1 and HeLa-EGFP were synchronized at the G1/S boundary by two cycles of thymidine blockage and at the G2/M boundary by nocodazole treatment. Briefly, exponentially growing cells were blocked for 16 h in 2 mM thymidine containing DMEM, released for 9 h in thymidine-free DMEM and blocked again with 2 mM thymidine containing DMEM for another 16 h. Synchronization at G2/M was achieved by culturing the cells with nocodazole (40 ng/ml) for 16 h. After the double-thymidine block and the removal of nocodazole, the cells were allowed to progress under normal conditions and harvested at different time points after release. The cell-cycle phase distribution of a cell population was determined by measuring cell DNA contents by flow cytometry as follows: cells (1 × 106) were trypsinized, washed and fixed in 75% ice-cold ethanol and stored at 4°C. Immediately before flow cytometric analysis, the samples were treated with RNase A for 30 min at room temperature and then stained with propidium iodide (final concentration 50 μg/ml) for 30 min at 37°C. The samples were analysed by using a BD LSR flow cytometer (Becton Dickinson, NJ). For immunofluorescence, cells were grown on glass coverslips and fixed with 4% paraformaldehyde for 20 min at room temperature at different time points after release. Cellular DNA was stained with 0.25 μg/ml 4′,6′-diamino-2-phenylindole (DAPI) for 10 min at room temperature. Coverslips were mounted with fluorescent mounting medium (DAKO) and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Jena, Germany).\n\nImmunostaining and confocal analysis\nDNA labelling and staining with 5-bromo-2′-deoxyuridine (BrdU labelling and detection kit I; Boehringer Mannheim, Germany) was performed according to the manufacturer's instructions. The nucleoli were detected with an anti-nucleolin antibody (clone 3G4B2; Upstate biotechnology, Lake Placid, NY) on cells fixed in 2% formalin for 10 min followed by permeabilization with ice-cold methanol for 30 min. The secondary antibody used for both BrdU and nucleoli staining was a rhodamine (tetra-methyl)-conjugated goat anti-mouse antibody (T-2762) from Molecular Probe (Eugene, OR). The cells were examined in a Zeiss LSM 510 laser scanning microscope equipped with a Plan-Apochromate 63×/1.4 oil immersion objective. We used the 488 nm laser line for excitation of EGFP (detected at 505 nm \u003c λEGFP \u003c 530 nm) and the 543 nm laser line for rhodamine (tetra-methyl) (detected at λRhodamine \u003e 560 nm). ECFP fusion protein was excited with a 458 nm laser line (detected at 480 nm \u003c λECFP \u003c 520 nm), EYFP fusion protein was excited with a 514 nm laser line (λEGFP \u003e 560 nm) and HcRed fusion protein was excited with a 543 nm laser line and detected at \u003e585 or 650 nm. The images were from 1 μm thick slices of the cells. The images were exported into Adobe Photoshop (Adobe Systems Inc., San Jose, CA).\n\nActinomycin D treatment\nFor actinomycin D treatment, HeLa-EGFP-hOGG1 cells were grown on chamber slides and synchronized at the G1/S boundary by two cycles of thymidine blockage. Cells were allowed to enter S-phase and actinomycin D was added 1 h after block release and incubated for 4 and 5 h, respectively. Cells were then washed twice with phosphate-buffered saline (PBS), fixed with 96% ethanol (anti-nucleolin) for 10 min at room temperature or 4% PFA (anti-fibrillarin and anti-RPA135) for 20 min and permeabilized with 0.5% Triton X-100 for 5 min at 4°C and washed again with PBS. Cells were air-dried before incubation with primary and secondary antibodies diluted in PBS with 1% BSA. As primary antibody monoclonal anti-nucleolin (clone 3G4B2; Upstate Biotechnology), RPA40 (N-20) (sc-17700; Santa Cruz), RPA135 (N-17) (sc-17913; Santa Cruz) and anti-fibrillarin (AFB01, cytoskeleton) were used and incubated overnight at room temperature. Incubation with secondary antibody (Alexa 594, 590/617; Molecular Probes) was carried out for 30 min at room temperature. DNA was stained with DAPI for 10 min at room temperature. Slides were mounted using the DAKO fluorescence mounting medium and examined using a Zeiss Axioplan 2 fluorescence microscope. Images were obtained using CoolSNAP (Photometrics) and then processed for publication using Jasc Pain Shop Pro (Jasc software).\n\nNuclear matrix preparation\nNuclear matrix proteins were fractionated from the indicated cells as described previously (47). For western blot analyses, equal cell equivalents from each fraction were subjected to 8% SDS–PAGE and probed with appropriate antibodies: rabbit polyclonal anti-GFP antibody (ab 290-50; Abcam), goat polyclonal anti-lamin A/C antibody (N-18; Santa Cruz), goat polyclonal anti-histone H1 antibody (C-17; Santa Cruz), monoclonal anti-human OGG1 antibody (clone 7E2; IBL) and rabbit polyclonal anti-phosphoserine antibody (poly-Z-PS1; Zymed Laboratories).\n","divisions":[{"label":"Title","span":{"begin":0,"end":21}},{"label":"Section","span":{"begin":149,"end":927}},{"label":"Title","span":{"begin":149,"end":183}},{"label":"Section","span":{"begin":929,"end":1738}},{"label":"Title","span":{"begin":929,"end":994}},{"label":"Section","span":{"begin":1740,"end":2448}},{"label":"Title","span":{"begin":1740,"end":1822}},{"label":"Section","span":{"begin":2450,"end":4095}},{"label":"Title","span":{"begin":2450,"end":2515}},{"label":"Section","span":{"begin":4097,"end":5424}},{"label":"Title","span":{"begin":4097,"end":4133}},{"label":"Section","span":{"begin":5426,"end":6810}},{"label":"Title","span":{"begin":5426,"end":5449}},{"label":"Section","span":{"begin":6812,"end":7388}},{"label":"Title","span":{"begin":6812,"end":6838}}],"tracks":[{"project":"2_test","denotations":[{"id":"15800211-10882850-76712973","span":{"begin":143,"end":145},"obj":"10882850"},{"id":"15800211-9321410-76712974","span":{"begin":277,"end":279},"obj":"9321410"},{"id":"15800211-12750383-76712975","span":{"begin":900,"end":902},"obj":"12750383"},{"id":"15800211-15200954-76712976","span":{"begin":909,"end":911},"obj":"15200954"},{"id":"15800211-10557315-76712977","span":{"begin":1958,"end":1960},"obj":"10557315"},{"id":"15800211-10882850-76712978","span":{"begin":2444,"end":2446},"obj":"10882850"},{"id":"15800211-12034821-76712979","span":{"begin":6931,"end":6933},"obj":"12034821"}],"attributes":[{"subj":"15800211-10882850-76712973","pred":"source","obj":"2_test"},{"subj":"15800211-9321410-76712974","pred":"source","obj":"2_test"},{"subj":"15800211-12750383-76712975","pred":"source","obj":"2_test"},{"subj":"15800211-15200954-76712976","pred":"source","obj":"2_test"},{"subj":"15800211-10557315-76712977","pred":"source","obj":"2_test"},{"subj":"15800211-10882850-76712978","pred":"source","obj":"2_test"},{"subj":"15800211-12034821-76712979","pred":"source","obj":"2_test"}]},{"project":"AnEM_full-texts","denotations":[{"id":"T1","span":{"begin":4321,"end":4329},"obj":"Cellular_component"},{"id":"T2","span":{"begin":4433,"end":4438},"obj":"Cell"},{"id":"T3","span":{"begin":4714,"end":4719},"obj":"Cell"},{"id":"T4","span":{"begin":5336,"end":5341},"obj":"Cell"},{"id":"T5","span":{"begin":4581,"end":4589},"obj":"Cellular_component"},{"id":"T6","span":{"begin":5322,"end":5328},"obj":"Cell"}],"attributes":[{"subj":"T1","pred":"source","obj":"AnEM_full-texts"},{"subj":"T2","pred":"source","obj":"AnEM_full-texts"},{"subj":"T3","pred":"source","obj":"AnEM_full-texts"},{"subj":"T4","pred":"source","obj":"AnEM_full-texts"},{"subj":"T5","pred":"source","obj":"AnEM_full-texts"},{"subj":"T6","pred":"source","obj":"AnEM_full-texts"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#e493ec","default":true},{"id":"AnEM_full-texts","color":"#93ecca"}]}]}}