sentences Home  

PMC:1310901 / 5129-13048 JSONTXT 18 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T34 0-21 Sentence denotes MATERIALS AND METHODS
T2707 23-33 Sentence denotes Cell lines
T35 23-33 Sentence denotes Cell lines
T2708 34-290 Sentence denotes K-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
T36 34-290 Sentence denotes K-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
T2709 291-362 Sentence denotes All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative.
T37 291-362 Sentence denotes All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative.
T2858 364-392 Sentence denotes Cell culture and stimulation
T38 364-392 Sentence denotes Cell culture and stimulation
T2859 393-611 Sentence denotes All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
T39 393-611 Sentence denotes All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
T2860 612-763 Sentence denotes When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods.
T40 612-763 Sentence denotes When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods.
T2861 764-842 Sentence denotes Owing to their chemical instability fresh substances were re-added every 24 h.
T41 764-842 Sentence denotes Owing to their chemical instability fresh substances were re-added every 24 h.
T3053 844-876 Sentence denotes Sequencing of the IRF-4 promoter
T42 844-876 Sentence denotes Sequencing of the IRF-4 promoter
T3054 877-1143 Sentence denotes For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended.
T43 877-1143 Sentence denotes For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended.
T3055 1144-1171 Sentence denotes The primers were 1-forward:
T44 1144-1171 Sentence denotes The primers were 1-forward:
T3056 1172-1212 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, 1-reverse:
T45 1172-1212 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, 1-reverse:
T3057 1213-1254 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′; 2-forward:
T46 1213-1254 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′; 2-forward:
T3058 1255-1298 Sentence denotes 5′-AGCGGTGAAACTGAGAGTGCGAGGT-3′, 2-reverse:
T47 1255-1298 Sentence denotes 5′-AGCGGTGAAACTGAGAGTGCGAGGT-3′, 2-reverse:
T3059 1299-1326 Sentence denotes 5′-GCCACATCGCTGCAGTTTAG-3′.
T48 1299-1326 Sentence denotes 5′-GCCACATCGCTGCAGTTTAG-3′.
T3060 1327-1424 Sentence denotes The products were cloned with the ‘TOPO TA cloning kit’ (Invitrogen, Groningen, The Netherlands).
T49 1327-1424 Sentence denotes The products were cloned with the ‘TOPO TA cloning kit’ (Invitrogen, Groningen, The Netherlands).
T3061 1425-1679 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.
T50 1425-1679 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.
T3408 1681-1700 Sentence denotes Expression analysis
T51 1681-1700 Sentence denotes Expression analysis
T3409 1701-1835 Sentence denotes To analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany).
T52 1701-1835 Sentence denotes To analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany).
T3410 1836-1922 Sentence denotes An aliquot of 1 μg total RNA was used for cDNA synthesis as described previously (27).
T53 1836-1922 Sentence denotes An aliquot of 1 μg total RNA was used for cDNA synthesis as described previously (27).
T3411 1923-2060 Sentence denotes RNA expression analysis for IRF-4 and the reference gene β-actin was carried out by semi-quantitative PCR as described previously (3,27).
T54 1923-2060 Sentence denotes RNA expression analysis for IRF-4 and the reference gene β-actin was carried out by semi-quantitative PCR as described previously (3,27).
T3412 2061-2112 Sentence denotes PCR products were verified by automated sequencing.
T55 2061-2112 Sentence denotes PCR products were verified by automated sequencing.
T3413 2113-2259 Sentence denotes PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28).
T56 2113-2259 Sentence denotes PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28).
T3414 2260-2377 Sentence denotes For analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29).
T57 2260-2377 Sentence denotes For analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29).
T3415 2378-2668 Sentence denotes Briefly, protein lysates were generated by incubating 1 × 106 cells in 100 µl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µl/ml protease-inhibitory-mix, 1 µmol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice.
T58 2378-2668 Sentence denotes Briefly, protein lysates were generated by incubating 1 × 106 cells in 100 µl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µl/ml protease-inhibitory-mix, 1 µmol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice.
T3416 2669-2799 Sentence denotes After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended.
T59 2669-2799 Sentence denotes After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended.
T3417 2800-2960 Sentence denotes Protein lysates (70–100 µg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 µm; Millipore, Eschborn, Germany).
T60 2800-2960 Sentence denotes Protein lysates (70–100 µg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 µm; Millipore, Eschborn, Germany).
T3418 2961-3338 Sentence denotes Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris–HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 µl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively.
T61 2961-3338 Sentence denotes Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris–HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 µl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively.
T3419 3339-3447 Sentence denotes The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany).
T62 3339-3447 Sentence denotes The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany).
T3420 3448-3587 Sentence denotes The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-β-actin (AC-74) (Sigma).
T63 3448-3587 Sentence denotes The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-β-actin (AC-74) (Sigma).
T4350 3589-3631 Sentence denotes Methylation-specific restriction-PCR-assay
T64 3589-3631 Sentence denotes Methylation-specific restriction-PCR-assay
T4351 3632-3696 Sentence denotes DNA was extracted with a commercial kit (Qiagen) as recommended.
T65 3632-3696 Sentence denotes DNA was extracted with a commercial kit (Qiagen) as recommended.
T4352 3697-3940 Sentence denotes Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24).
T66 3697-3940 Sentence denotes Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24).
T4353 3941-4070 Sentence denotes As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used.
T67 3941-4070 Sentence denotes As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used.
T4354 4071-4194 Sentence denotes DNA (0.8 µg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h.
T68 4071-4194 Sentence denotes DNA (0.8 µg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h.
T4355 4195-4363 Sentence denotes Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A).
T69 4195-4363 Sentence denotes Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A).
T4356 4364-4409 Sentence denotes The sequences of the primers were F1-forward:
T70 4364-4409 Sentence denotes The sequences of the primers were F1-forward:
T4357 4410-4502 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3′ (341 bp); F2-forward:
T71 4410-4502 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3′ (341 bp); F2-forward:
T4358 4503-4547 Sentence denotes 5′-AAGGTGAACTGAAGTCTGGAAGTGA-3′, F2-reverse:
T72 4503-4547 Sentence denotes 5′-AAGGTGAACTGAAGTCTGGAAGTGA-3′, F2-reverse:
T4359 4548-4587 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′ (474 bp).
T73 4548-4587 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′ (474 bp).
T4360 4588-4636 Sentence denotes The PCR conditions were described elsewhere (3).
T74 4588-4636 Sentence denotes The PCR conditions were described elsewhere (3).
T4361 4637-4707 Sentence denotes PCR was performed with an annealing temperature of 62°C and 35 cycles.
T75 4637-4707 Sentence denotes PCR was performed with an annealing temperature of 62°C and 35 cycles.
T4362 4708-4908 Sentence denotes When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated.
T76 4708-4908 Sentence denotes When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated.
T4363 4909-5020 Sentence denotes The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed.
T77 4909-5020 Sentence denotes The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed.
T4364 5021-5072 Sentence denotes PCR products were verified by automated sequencing.
T78 5021-5072 Sentence denotes PCR products were verified by automated sequencing.
T4932 5074-5093 Sentence denotes Bisulfite treatment
T79 5074-5093 Sentence denotes Bisulfite treatment
T4933 5094-5131 Sentence denotes DNA was extracted as described above.
T80 5094-5131 Sentence denotes DNA was extracted as described above.
T4934 5132-5311 Sentence denotes Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows.
T81 5132-5311 Sentence denotes Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows.
T4935 5312-5480 Sentence denotes Briefly, 1 µg of DNA and 2 µg of poly(dA–dT)(poly(dA–dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42°C in 0.3 M NaOH in a volume of 50 µl.
T82 5312-5480 Sentence denotes Briefly, 1 µg of DNA and 2 µg of poly(dA–dT)(poly(dA–dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42°C in 0.3 M NaOH in a volume of 50 µl.
T83 5481-5702 Sentence denotes Fresh solutions of 30 µl of 10 mM hydrochinon (Sigma) and 530 µl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12–13 h at 50°C.
T4936 5481-5803 Sentence denotes Fresh solutions of 30 µl of 10 mM hydrochinon (Sigma) and 530 µl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12–13 h at 50°C. The aqueous phase was recovered using the ‘Wizard DNA clean-up system’ (Promega, Mannheim, Germany).
T84 5703-5803 Sentence denotes The aqueous phase was recovered using the ‘Wizard DNA clean-up system’ (Promega, Mannheim, Germany).
T4937 5804-5960 Sentence denotes The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37°C to ensure complete desulfonisation.
T85 5804-5960 Sentence denotes The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37°C to ensure complete desulfonisation.
T4938 5961-6094 Sentence denotes DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 µl H2O.
T86 5961-6094 Sentence denotes DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 µl H2O.
T4939 6095-6310 Sentence denotes Subsequent PCR amplification of 4 µl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the ‘TOPO TA cloning kit’ (Invitrogen) (see Figure 3A).
T87 6095-6310 Sentence denotes Subsequent PCR amplification of 4 µl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the ‘TOPO TA cloning kit’ (Invitrogen) (see Figure 3A).
T4940 6311-6734 Sentence denotes The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5′-TATTTGGATTTTTAGGGAGTTTTTTTT-3′, BS-I-reverse 5′-ACCCAACTCCCTTAAACTATTAAACT-3′ (187 bp); BS-II-forward 5′-AGTTTAATAGTTTAAGGGAGTTGGGT-3′, BS-II-reverse 5′-CTCACCCTAAACTCAAAACTAAAAAC-3′ (674 bp).
T88 6311-6734 Sentence denotes The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5′-TATTTGGATTTTTAGGGAGTTTTTTTT-3′, BS-I-reverse 5′-ACCCAACTCCCTTAAACTATTAAACT-3′ (187 bp); BS-II-forward 5′-AGTTTAATAGTTTAAGGGAGTTGGGT-3′, BS-II-reverse 5′-CTCACCCTAAACTCAAAACTAAAAAC-3′ (674 bp).
T4941 6735-6926 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems).
T89 6735-6926 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems).
T5684 6928-6973 Sentence denotes In vitro methylation and reporter gene assays
T90 6928-6973 Sentence denotes In vitro methylation and reporter gene assays
T91 6974-7046 Sentence denotes The IRF-4 promoter-reporter gene construct was generously provided by J.
T5685 6974-7060 Sentence denotes The IRF-4 promoter-reporter gene construct was generously provided by J. Hiscott (31).
T92 7047-7060 Sentence denotes Hiscott (31).
T5686 7061-7248 Sentence denotes Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A).
T93 7061-7248 Sentence denotes Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A).
T5687 7249-7360 Sentence denotes Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29).
T94 7249-7360 Sentence denotes Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29).
T5688 7361-7528 Sentence denotes Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation.
T95 7361-7528 Sentence denotes Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation.
T5689 7529-7611 Sentence denotes The control construct served as an internal reference for transfection efficiency.
T96 7529-7611 Sentence denotes The control construct served as an internal reference for transfection efficiency.
T5690 7612-7747 Sentence denotes Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany).
T97 7612-7747 Sentence denotes Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany).
T5691 7748-7864 Sentence denotes IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu).
T98 7748-7864 Sentence denotes IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu).
T5692 7865-7918 Sentence denotes Each experiment was carried out at least three times.
T99 7865-7918 Sentence denotes Each experiment was carried out at least three times.