uvrd dna helicase Search Results


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  • 99
    New England Biolabs t4 polynucleotide kinase
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher total rna
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 471882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology recq1
    Kinetics of FEN-1 cleavage of the 15nt 5’-flap DNA substrate in the presence or absence of <t>RECQ1</t> Reactions (200 μl) containing 10 fmol of 15 nt 5’-flap DNA substrate and 0.3 nM of FEN-1 with or without RECQ1 (1 nM) were incubated at 37°C and 20 μl aliquots were removed at indicated time points. A. Phosphorimage of a typical gel from a time course experiment. Increasing times of incubation (0-24 min) for the FEN-1 cleavage reactions conducted in the absence of RECQ1 (lanes 2-10) or in the presence of RECQ1 (lanes 11-19) are indicated. No enzyme and RECQ1 alone reactions are indicated in lane 1 and 20, respectively. B. Percent incision from the data shown in ‘A’, data points are the mean of three independent experiments with SD indicated by error bars.
    Recq1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene recq1
    Nucleolar <t>RECQ1</t> undergoes re-localization to chromatin in response to IR. Indirect immunofluorescence was performed on HeLa cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure as described in “ Materials and Methods ”. Panel A , Nucleolar localization of RECQ1 in untreated cells. Cells were co-immunostained with anti-RECQ1 and anti-nucleolin. The merged images show cells stained with RECQ1 (red) and nucleolin (green), with or without DAPI (blue). Panel B, Relocalization of RECQ1 to chromatin-bound foci following IR damage. After IR exposure, cells were stained for total RECQ1 (top panel) or in situ detergent extraction-resistant RECQ1 (middle and bottom panel). Merged images show RECQ1 (red) and DAPI (blue). Co-immunostaining of chromatin-bound RECQ1 and γ-H2AX ( Panel B, bottom ). Merged images show RECQ1 (red) and γ-H2AX (green), with or without DAPI (blue). Panel C, Schematic presentation of the protocol for sequential nuclear fractionation of lysates from U2OS cells either untreated or following 6 h recovery from 10 Gy IR exposure. Cytoplasmic and nucleoplasmic proteins were extracted by permeabilization with detergent, and the resulting nuclei were nuclease-digested and extracted with NH 2 SO 4 . Proteins of the supernatant (S) and pellet (P) fractions were resolved on 10% SDS-PAGE, and subsequently analyzed by Western blot for RECQ1, histone H4 (chromatin marker), and lamin B (nuclear matrix marker). Panel D, Following IR treatment, RECQ1 is enriched in the chromatin fraction (S4).
    Recq1, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    ATUM dna replication helicase nuclease 2
    Hub modules from the PPI network in osteoarthritis patients were constructed. (A) A total of five upregulated proteins constituted a hub PPI module: GOSR1, SEC22C, VAMP7, STX16) and VAMP3. (B and C) Twelve downregulated proteins constituted 2 hub PPI modules: CCL5, GAL, CXCL5, APP, ADCY9, HRH4, CXCR5, RPA1, RAD50, DNA2, RAD17 and RECQL5. PPI, protein-protein interaction. GOSR1, golgi SNAP receptor complex member 1; SEC22C, SEC22 homolog C vesicle trafficking protein; VAMP7, vesicle associated membrane protein 7; STX16, syntaxin 16; VAMP3, vesicle associated membrane protein 3; CCL5, C-C motif chemokine ligand 5; GAL, galanin and GMAP prepropeptide; CXCL5, C-X-C motif chemokine ligand 5; APP, amyloid beta precursor protein; ADCY9, adenylate cyclase 9; HRH4, histamine receptor H4; CXCR5, C-X-C motif chemokine receptor 5; RPA1, replication protein A1; RAD50, RAD50 double strand break repair protein; DNA2, <t>DNA</t> replication <t>helicase/nuclease</t> 2; RAD17, RAD17 checkpoint clamp loader component; RECQL5, RecQ like helicase 5.
    Dna Replication Helicase Nuclease 2, supplied by ATUM, used in various techniques. Bioz Stars score: 89/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Double Helix dna double helix
    The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder <t>distamycin</t> A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex <t>DNA</t> substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.
    Dna Double Helix, supplied by Double Helix, used in various techniques. Bioz Stars score: 94/100, based on 10708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Pacific Biosciences ec1515
    The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder <t>distamycin</t> A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex <t>DNA</t> substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.
    Ec1515, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cell Signaling Technology Inc anti rig i
    Human immature moDCs constantly express NLRC5, NLRX1, <t>RIG-I</t> and MDA5. (A–D) Freshly isolated monocytes were seeded in 24-well plates and differentiated as described in the “Materials and Methods.” The protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were measured by western blot. (A,C) Representative blots are shown. (B,D) Graphs represent the kinetics of protein expressions during moDC differentiation. Data are represented as mean ± SD of 3–5 individual experiments and were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p
    Anti Rig I, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad polyacrylamide gels
    Human immature moDCs constantly express NLRC5, NLRX1, <t>RIG-I</t> and MDA5. (A–D) Freshly isolated monocytes were seeded in 24-well plates and differentiated as described in the “Materials and Methods.” The protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were measured by western blot. (A,C) Representative blots are shown. (B,D) Graphs represent the kinetics of protein expressions during moDC differentiation. Data are represented as mean ± SD of 3–5 individual experiments and were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p
    Polyacrylamide Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 13598 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Abcam recql5
    Intercalating agents affect the recruitment of <t>RECQL5</t> to the sites of ICLs. U2OS cells were transfected with either 2 µg GFP–RECQL5 ( A ) or 2 µg GFP-XPC ( D ). Twenty-four hours posttransfection, the cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( B ) U2OS cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for γ-H2AX (red) to indicate a damage signal. The targeted regions are indicated by arrows. U2OS cells were transfected with either 2 µg GFP-RECQL5 ( C ) or 2 µg GFP-XPC ( E ). Twenty-four hours posttransfection, the cells were incubated with EtBr at 5 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.
    Recql5, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam ku70 80
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Ku70 80, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ku80
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Ku80, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad edta
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Edta, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Junsei Chemical mgcl2
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Mgcl2, supplied by Junsei Chemical, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad pcr amplification
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Pcr Amplification, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 13424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Aragon smc5 6 complex
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Smc5 6 Complex, supplied by Aragon, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iscript cdna synthesis kit
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    Iscript Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 95548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad pharosfx imager
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Journal of Biological Chemistry chemistry 2008
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Thermo Fisher ct 1 step kit
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Millipore atp disodium
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Millipore atp
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Double Helix dna double helix chain
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or <t>Ku70/80</t> (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
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    Thermo Fisher gene exp chd7 hs00215010 m1
    Transfection with <t>CHD7</t> dominant-negative (DN) mRNA transcripts disrupted differentiation potential and cell proliferation of ESCs. ( A ) Protocol for transfection with CHD7 ). Transcripts were transfected into KhES-1 cells on day 0, and the cells were then transferred to low-attachment plates for 24 h, followed by culture in Es6 medium with Rock Inhibitor (RI) for 24 h for EB formation. Microscopic observation of EBs and gene expression profiles by qRT-PCR scorecard panel on day 3 after transfection with DN mRNA. ( B ) CHD7 DN 1 and CHD7 DN2 expression levels were determined by qRT-PCR. A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of day 3-EBs from non-transfected, CHD7 DN1-, CHD7 DN2-, CHD7 -DN1 + CHD7 -DN2-transfected, and mock mRNA-transfected KhES-1 3 days after transfection. Gene expression profiles were determined using qRT-PCR scorecard panels and are shown below the relevant photograph. The cell number on day 3 of culture in one well of a 6-well plate was scored and appended at the top right corner of the relevant image. Non-transfected KhES-1 cultured with Es8 on day 0 (left panel) was used as a control. Representative results of 3 independent experiments are shown. Scale bar: 1 mm.
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    Image Search Results


    Kinetics of FEN-1 cleavage of the 15nt 5’-flap DNA substrate in the presence or absence of RECQ1 Reactions (200 μl) containing 10 fmol of 15 nt 5’-flap DNA substrate and 0.3 nM of FEN-1 with or without RECQ1 (1 nM) were incubated at 37°C and 20 μl aliquots were removed at indicated time points. A. Phosphorimage of a typical gel from a time course experiment. Increasing times of incubation (0-24 min) for the FEN-1 cleavage reactions conducted in the absence of RECQ1 (lanes 2-10) or in the presence of RECQ1 (lanes 11-19) are indicated. No enzyme and RECQ1 alone reactions are indicated in lane 1 and 20, respectively. B. Percent incision from the data shown in ‘A’, data points are the mean of three independent experiments with SD indicated by error bars.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: Kinetics of FEN-1 cleavage of the 15nt 5’-flap DNA substrate in the presence or absence of RECQ1 Reactions (200 μl) containing 10 fmol of 15 nt 5’-flap DNA substrate and 0.3 nM of FEN-1 with or without RECQ1 (1 nM) were incubated at 37°C and 20 μl aliquots were removed at indicated time points. A. Phosphorimage of a typical gel from a time course experiment. Increasing times of incubation (0-24 min) for the FEN-1 cleavage reactions conducted in the absence of RECQ1 (lanes 2-10) or in the presence of RECQ1 (lanes 11-19) are indicated. No enzyme and RECQ1 alone reactions are indicated in lane 1 and 20, respectively. B. Percent incision from the data shown in ‘A’, data points are the mean of three independent experiments with SD indicated by error bars.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Incubation

    RECQ1 associates with telomere chromatin and stimulates FEN-1 cleavage of 5’-flap containing telomeric repeat sequence A. ChIP-qPCR of immunoprecipitated DNA with probes specific for telomeric region. HeLa cells were processed for ChIP using a RECQ1-specific antibody. FEN-1 antibody was used as a positive control for telomere enrichment and rabbit IgG served as negative control in ChIP experiments. Quantification of cross-linked telomere chromatin immunoprecipitated using the indicated antibodies is shown. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Relative occupancy at telomere versus a non-telomere negative control site (DNA containing HBG) and GAPDH shows preferential association of RECQ1 to telomeres. Results are expressed as means ± SEM for at least three independent experiments. B .A representative gel of the amplified telomere DNA immunoprecipitated with RECQ1 antibody. PCR amplified telomere fragments migrated as a smear (50 to ~500 bp). C. Phosphorimage of a typical gel of RECQ1 stimulation of FEN-1 incision of a 15 nt 5’-flap substrate containing TTAGGG repeats in duplex region upstream of 5’-flap. D. Percent incision of 15 nt 5’-flap substrate containing non-telomeric (solid line) or telomeric (dashed line) sequence. Data indicates mean of at least three independent experiments with SD shown as error bars. E. Phosphorimage of a typical gel of RECQ1 (0-2 nM) stimulation of FEN-1 incision of 5’-flap substrates containing non telomeric sequence or TTAGGG repeats in the 5’-flap. F. Percent incision of 5’-flap substrate containing non-telomeric (solid line) or telomeric (dashed line) sequence in the 5’-flap. Data indicates mean of at least three independent experiments with SD shown as error bars.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: RECQ1 associates with telomere chromatin and stimulates FEN-1 cleavage of 5’-flap containing telomeric repeat sequence A. ChIP-qPCR of immunoprecipitated DNA with probes specific for telomeric region. HeLa cells were processed for ChIP using a RECQ1-specific antibody. FEN-1 antibody was used as a positive control for telomere enrichment and rabbit IgG served as negative control in ChIP experiments. Quantification of cross-linked telomere chromatin immunoprecipitated using the indicated antibodies is shown. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Relative occupancy at telomere versus a non-telomere negative control site (DNA containing HBG) and GAPDH shows preferential association of RECQ1 to telomeres. Results are expressed as means ± SEM for at least three independent experiments. B .A representative gel of the amplified telomere DNA immunoprecipitated with RECQ1 antibody. PCR amplified telomere fragments migrated as a smear (50 to ~500 bp). C. Phosphorimage of a typical gel of RECQ1 stimulation of FEN-1 incision of a 15 nt 5’-flap substrate containing TTAGGG repeats in duplex region upstream of 5’-flap. D. Percent incision of 15 nt 5’-flap substrate containing non-telomeric (solid line) or telomeric (dashed line) sequence. Data indicates mean of at least three independent experiments with SD shown as error bars. E. Phosphorimage of a typical gel of RECQ1 (0-2 nM) stimulation of FEN-1 incision of 5’-flap substrates containing non telomeric sequence or TTAGGG repeats in the 5’-flap. F. Percent incision of 5’-flap substrate containing non-telomeric (solid line) or telomeric (dashed line) sequence in the 5’-flap. Data indicates mean of at least three independent experiments with SD shown as error bars.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Positive Control, Negative Control, Amplification, Polymerase Chain Reaction

    FEN-1 binding activity of RECQ1 is contained within RQC and extreme C-terminal end A. Schematic representation of GST-RECQ1 recombinant fragments used for FEN-1 pull-down experiments. B. Coomassie-stained 10% SDS-PAGE gel showing purified FEN-1 used for binding assays. C. Ponceau S-stained membrane showing protein complexes bound to glutathione sepharose beads in pull-down assay. Beads were mixed with lysate from bacteria expressing GST fusion proteins containing human RECQ1 fragments or GST alone as indicated. D. Purified recombinant FEN-1 protein (200 ng) was added to the indicated GST-RECQ1 bound glutathione sepharose beads. After washing, protein complexes were eluted and resolved by SDS-PAGE. Bound FEN-1 was detected by Western blotting.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: FEN-1 binding activity of RECQ1 is contained within RQC and extreme C-terminal end A. Schematic representation of GST-RECQ1 recombinant fragments used for FEN-1 pull-down experiments. B. Coomassie-stained 10% SDS-PAGE gel showing purified FEN-1 used for binding assays. C. Ponceau S-stained membrane showing protein complexes bound to glutathione sepharose beads in pull-down assay. Beads were mixed with lysate from bacteria expressing GST fusion proteins containing human RECQ1 fragments or GST alone as indicated. D. Purified recombinant FEN-1 protein (200 ng) was added to the indicated GST-RECQ1 bound glutathione sepharose beads. After washing, protein complexes were eluted and resolved by SDS-PAGE. Bound FEN-1 was detected by Western blotting.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Binding Assay, Activity Assay, Recombinant, Staining, SDS Page, Purification, Pull Down Assay, Expressing, Western Blot

    Mapping of the FEN-1 interaction domains that mediate the functional interaction between RECQ1 and FEN-1 Reactions (20 μl) containing 10 fmol of 15nt 5’-flap DNA substrate, 0.3 nM of FEN-1 and indicated concentrations of GST or GST-fused RECQ1, full-length (FL), helicase domain (HD), RecQ-C-terminus domain (RQC), or C-terminal (Ct), were incubated at 37°C for 15 min under standard conditions. A. Phosphorimage of a typical gel. Lane 1, no enzyme; B. Percent incision from the mean of three independent experiments is shown with SD indicated by error bars.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: Mapping of the FEN-1 interaction domains that mediate the functional interaction between RECQ1 and FEN-1 Reactions (20 μl) containing 10 fmol of 15nt 5’-flap DNA substrate, 0.3 nM of FEN-1 and indicated concentrations of GST or GST-fused RECQ1, full-length (FL), helicase domain (HD), RecQ-C-terminus domain (RQC), or C-terminal (Ct), were incubated at 37°C for 15 min under standard conditions. A. Phosphorimage of a typical gel. Lane 1, no enzyme; B. Percent incision from the mean of three independent experiments is shown with SD indicated by error bars.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Functional Assay, Incubation

    RECQ1 binding activity of FEN-1 is contained within amino acids 328-380 A. Schematic representation of GST-FEN-1 recombinant fragments used for pull-down experiments. B. Coomassie-stained 10% SDS-PAGE gel showing purified RECQ1. C. Ponceau S-stained membrane showing protein complexes bound to glutathione sepharose beads in pull-down assay. Beads were mixed with lysate from bacteria expressing GST fusion proteins containing human FEN-1 fragments or GST alone as indicated. D. Purified recombinant RECQ1 protein (200 ng) was added to the indicated GST-FEN-1 bound glutathione sepharose beads. After washing, protein complexes were eluted and resolved by SDS-PAGE. Bound RECQ1 was detected by Western blotting.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: RECQ1 binding activity of FEN-1 is contained within amino acids 328-380 A. Schematic representation of GST-FEN-1 recombinant fragments used for pull-down experiments. B. Coomassie-stained 10% SDS-PAGE gel showing purified RECQ1. C. Ponceau S-stained membrane showing protein complexes bound to glutathione sepharose beads in pull-down assay. Beads were mixed with lysate from bacteria expressing GST fusion proteins containing human FEN-1 fragments or GST alone as indicated. D. Purified recombinant RECQ1 protein (200 ng) was added to the indicated GST-FEN-1 bound glutathione sepharose beads. After washing, protein complexes were eluted and resolved by SDS-PAGE. Bound RECQ1 was detected by Western blotting.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Binding Assay, Activity Assay, Recombinant, Staining, SDS Page, Purification, Pull Down Assay, Expressing, Western Blot

    RECQ1 stimulates FEN-1 cleavage of 5’-flap DNA substrate Reactions (20 μl) containing 10 fmol DNA substrate, indicted amounts of FEN-1 and increasing concentration of RECQ1 (0-2.0 nM) were incubated at 37°C for 15 min under conditions described in Experimental methods. Star indicates position of 32 P label. A. Phosphorimage of a typical gel of FEN-1 incision activity on a 15 nt 5’-flap DNA substrate. B. Percent incision from the data shown in ‘A’, data points are the mean of three independent experiments with SDs indicated by error bars. C. RECQ1 stimulation of FEN-1 cleavage of 5’-flap substrates with increasing length of 5’-flap (1, 5, 15, and 26 nt). Percent incision by FEN-1 is shown as the mean of three independent experiments with SD indicated by error bars. D. FEN-1 stimulation by PCNA or RECQ1 is mutually exclusive. Incision reactions, performed as above, contained either FEN-1 alone, or the indicated amounts of PCNA or /and RECQ1. E. Percent incision from the representative experiment shown in ‘D’, data points are the mean of three independent experiments with SDs indicated by error bars.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: RECQ1 stimulates FEN-1 cleavage of 5’-flap DNA substrate Reactions (20 μl) containing 10 fmol DNA substrate, indicted amounts of FEN-1 and increasing concentration of RECQ1 (0-2.0 nM) were incubated at 37°C for 15 min under conditions described in Experimental methods. Star indicates position of 32 P label. A. Phosphorimage of a typical gel of FEN-1 incision activity on a 15 nt 5’-flap DNA substrate. B. Percent incision from the data shown in ‘A’, data points are the mean of three independent experiments with SDs indicated by error bars. C. RECQ1 stimulation of FEN-1 cleavage of 5’-flap substrates with increasing length of 5’-flap (1, 5, 15, and 26 nt). Percent incision by FEN-1 is shown as the mean of three independent experiments with SD indicated by error bars. D. FEN-1 stimulation by PCNA or RECQ1 is mutually exclusive. Incision reactions, performed as above, contained either FEN-1 alone, or the indicated amounts of PCNA or /and RECQ1. E. Percent incision from the representative experiment shown in ‘D’, data points are the mean of three independent experiments with SDs indicated by error bars.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Concentration Assay, Incubation, Activity Assay

    Stimulation of FEN-1 cleavage of 5’-flap is independent of RECQ1 helicase activity and FEN-1 does not alter RECQ1 helicase activity A. FEN-1 does not alter RECQ1 helicase activity on a fork duplex. Phosphorimage of a typical helicase gel showing unwinding of a fork duplex by RECQ1 in the presence or absence of FEN-1; heat denatured substrate is indicated by triangle (lane 8). B. Reactions (20 μl) containing 10 fmol of 15nt 5’-flap DNA substrate and indicated concentrations of FEN-1 and/or RECQ1, wild-type (WT) or helicase dead mutant (K119A), proteins were incubated at 37°C for 15 min under standard conditions. Phosphorimage of a typical gel shows helicase-dead mutant of RECQ1 stimulates FEN-1 cleavage of 15nt 5’-flap DNA substrate. Lane 1, no enzyme; lane 2-7, FEN-1+ RECQ1 wild-type (0-2 nM); lane 8-13, FEN-1 + RECQ1 K119A mutant (0-2 nM). C. RECQ1 stimulates FEN-1 activity on 15nt 5’-flap DNA substrate in the absence of ATP. Phosphorimage of a typical gel shows FEN-1 incision products from reactions performed in the presence or absence of ATP.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: Stimulation of FEN-1 cleavage of 5’-flap is independent of RECQ1 helicase activity and FEN-1 does not alter RECQ1 helicase activity A. FEN-1 does not alter RECQ1 helicase activity on a fork duplex. Phosphorimage of a typical helicase gel showing unwinding of a fork duplex by RECQ1 in the presence or absence of FEN-1; heat denatured substrate is indicated by triangle (lane 8). B. Reactions (20 μl) containing 10 fmol of 15nt 5’-flap DNA substrate and indicated concentrations of FEN-1 and/or RECQ1, wild-type (WT) or helicase dead mutant (K119A), proteins were incubated at 37°C for 15 min under standard conditions. Phosphorimage of a typical gel shows helicase-dead mutant of RECQ1 stimulates FEN-1 cleavage of 15nt 5’-flap DNA substrate. Lane 1, no enzyme; lane 2-7, FEN-1+ RECQ1 wild-type (0-2 nM); lane 8-13, FEN-1 + RECQ1 K119A mutant (0-2 nM). C. RECQ1 stimulates FEN-1 activity on 15nt 5’-flap DNA substrate in the absence of ATP. Phosphorimage of a typical gel shows FEN-1 incision products from reactions performed in the presence or absence of ATP.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: Activity Assay, Mutagenesis, Incubation

    RECQ1 interacts with FEN-1 in vivo and in vitro A. Co-IP analysis of RECQ1 interaction with FEN-1 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, FEN-1 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained FEN-1. Reciprocal co-IPs of FEN-1 also contained RECQ1. FEN-1 antibody also detected Ig heavy and light chains ( indicated by asterisks ) in IP-Western and interfered with FEN-1 signal (~ 42 kD). B. Association of RECQ1 and FEN-1 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and FEN-1 using benzonase-treated extract in IP reaction. Reciprocal co-IPs of FEN-1 also contained RECQ1. C. Reciprocal co-IP of RECQ1 and FEN-1 from benzonase-treated extracts prepared from HeLa cells transduced with lentiviral-expressed RNA interference (RNAi) hairpins (shRNA) targeting RECQ1 or luciferase (negative control, CTL). D. Recombinant RECQ1 and FEN-1 proteins directly interact in vitro as shown by ELISA. Either BSA or purified recombinant RECQ1 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant FEN-1 (0-50 nM) for 1 h at 30°C. Following washing, RECQ1-bound FEN-1 was detected by ELISA using anti-FEN-1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars.

    Journal: The Biochemical journal

    Article Title: RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    doi: 10.1042/BJ20141021

    Figure Lengend Snippet: RECQ1 interacts with FEN-1 in vivo and in vitro A. Co-IP analysis of RECQ1 interaction with FEN-1 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, FEN-1 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained FEN-1. Reciprocal co-IPs of FEN-1 also contained RECQ1. FEN-1 antibody also detected Ig heavy and light chains ( indicated by asterisks ) in IP-Western and interfered with FEN-1 signal (~ 42 kD). B. Association of RECQ1 and FEN-1 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and FEN-1 using benzonase-treated extract in IP reaction. Reciprocal co-IPs of FEN-1 also contained RECQ1. C. Reciprocal co-IP of RECQ1 and FEN-1 from benzonase-treated extracts prepared from HeLa cells transduced with lentiviral-expressed RNA interference (RNAi) hairpins (shRNA) targeting RECQ1 or luciferase (negative control, CTL). D. Recombinant RECQ1 and FEN-1 proteins directly interact in vitro as shown by ELISA. Either BSA or purified recombinant RECQ1 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant FEN-1 (0-50 nM) for 1 h at 30°C. Following washing, RECQ1-bound FEN-1 was detected by ELISA using anti-FEN-1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars.

    Article Snippet: Here, we report a direct protein interaction between RECQ1 and FEN-1, and demonstrate that RECQ1 stimulates FEN-1 cleavage of a 5’-flap DNA substrate independent of its helicase activity.

    Techniques: In Vivo, In Vitro, Co-Immunoprecipitation Assay, Western Blot, Transduction, shRNA, Luciferase, Negative Control, CTL Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Incubation

    Nucleolar RECQ1 undergoes re-localization to chromatin in response to IR. Indirect immunofluorescence was performed on HeLa cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure as described in “ Materials and Methods ”. Panel A , Nucleolar localization of RECQ1 in untreated cells. Cells were co-immunostained with anti-RECQ1 and anti-nucleolin. The merged images show cells stained with RECQ1 (red) and nucleolin (green), with or without DAPI (blue). Panel B, Relocalization of RECQ1 to chromatin-bound foci following IR damage. After IR exposure, cells were stained for total RECQ1 (top panel) or in situ detergent extraction-resistant RECQ1 (middle and bottom panel). Merged images show RECQ1 (red) and DAPI (blue). Co-immunostaining of chromatin-bound RECQ1 and γ-H2AX ( Panel B, bottom ). Merged images show RECQ1 (red) and γ-H2AX (green), with or without DAPI (blue). Panel C, Schematic presentation of the protocol for sequential nuclear fractionation of lysates from U2OS cells either untreated or following 6 h recovery from 10 Gy IR exposure. Cytoplasmic and nucleoplasmic proteins were extracted by permeabilization with detergent, and the resulting nuclei were nuclease-digested and extracted with NH 2 SO 4 . Proteins of the supernatant (S) and pellet (P) fractions were resolved on 10% SDS-PAGE, and subsequently analyzed by Western blot for RECQ1, histone H4 (chromatin marker), and lamin B (nuclear matrix marker). Panel D, Following IR treatment, RECQ1 is enriched in the chromatin fraction (S4).

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Nucleolar RECQ1 undergoes re-localization to chromatin in response to IR. Indirect immunofluorescence was performed on HeLa cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure as described in “ Materials and Methods ”. Panel A , Nucleolar localization of RECQ1 in untreated cells. Cells were co-immunostained with anti-RECQ1 and anti-nucleolin. The merged images show cells stained with RECQ1 (red) and nucleolin (green), with or without DAPI (blue). Panel B, Relocalization of RECQ1 to chromatin-bound foci following IR damage. After IR exposure, cells were stained for total RECQ1 (top panel) or in situ detergent extraction-resistant RECQ1 (middle and bottom panel). Merged images show RECQ1 (red) and DAPI (blue). Co-immunostaining of chromatin-bound RECQ1 and γ-H2AX ( Panel B, bottom ). Merged images show RECQ1 (red) and γ-H2AX (green), with or without DAPI (blue). Panel C, Schematic presentation of the protocol for sequential nuclear fractionation of lysates from U2OS cells either untreated or following 6 h recovery from 10 Gy IR exposure. Cytoplasmic and nucleoplasmic proteins were extracted by permeabilization with detergent, and the resulting nuclei were nuclease-digested and extracted with NH 2 SO 4 . Proteins of the supernatant (S) and pellet (P) fractions were resolved on 10% SDS-PAGE, and subsequently analyzed by Western blot for RECQ1, histone H4 (chromatin marker), and lamin B (nuclear matrix marker). Panel D, Following IR treatment, RECQ1 is enriched in the chromatin fraction (S4).

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Immunofluorescence, Staining, In Situ, Immunostaining, Fractionation, SDS Page, Western Blot, Marker

    Effect of RECQ1 depletion on G2/M and intra-S-phase DNA damage checkpoints. Panel A , DNA damage-induced mitotic entry delay is minimally affected by the RECQ1 deficiency. HeLa cells were transfected with the indicated siRNAs and either mock-treated or exposed to 3 Gy of IR 1 h before harvesting. Mitotic cells were detected by PI and phosphohistone H3 staining and analyzed by flow cytometry. Percentages of mitotic cells and their levels normalized to control (in parentheses) are shown. Panel B, RECQ1 is involved in maintenance of IR-induced G2/M checkpoint. Cells with either control or RECQ1 siRNA were exposed to 10 Gy of IR. Nocodazole (1 µg/ml) was added to the medium at the time of IR treatment to capture cells entering mitosis. 16 h later, cells were collected for PI and phospho-histone H3 staining and analyzed by flow cytometry. Panel C, HeLa cells transfected with control or RECQ1 siRNAs were exposed to 10 Gy of IR and assayed for DNA synthesis 30 min later by [ 3 H]thymidine incorporation. The amount of DNA synthesis after irradiation is expressed as a percentage of the level in untreated cells. Error bars indicate SD from three independent experiments.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Effect of RECQ1 depletion on G2/M and intra-S-phase DNA damage checkpoints. Panel A , DNA damage-induced mitotic entry delay is minimally affected by the RECQ1 deficiency. HeLa cells were transfected with the indicated siRNAs and either mock-treated or exposed to 3 Gy of IR 1 h before harvesting. Mitotic cells were detected by PI and phosphohistone H3 staining and analyzed by flow cytometry. Percentages of mitotic cells and their levels normalized to control (in parentheses) are shown. Panel B, RECQ1 is involved in maintenance of IR-induced G2/M checkpoint. Cells with either control or RECQ1 siRNA were exposed to 10 Gy of IR. Nocodazole (1 µg/ml) was added to the medium at the time of IR treatment to capture cells entering mitosis. 16 h later, cells were collected for PI and phospho-histone H3 staining and analyzed by flow cytometry. Panel C, HeLa cells transfected with control or RECQ1 siRNAs were exposed to 10 Gy of IR and assayed for DNA synthesis 30 min later by [ 3 H]thymidine incorporation. The amount of DNA synthesis after irradiation is expressed as a percentage of the level in untreated cells. Error bars indicate SD from three independent experiments.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Transfection, Staining, Flow Cytometry, Cytometry, DNA Synthesis, Irradiation

    RECQ1 depletion sensitizes cells to DNA damage induced by ionizing radiation or camptothecin. siRNA knockdown of RECQ1 in HeLa cells leads to increased sensitivity to treatment with IR ( Panel A ) or CPT ( Panel B ). HeLa cells treated with either RECQ1 siRNA or control siRNA (#C) were plated in quadruplicate and treated with increasing doses of IR or concentrations of CPT. Total DNA content was measured as an indication of cell growth. Two independent siRNAs (#L1 and #L2) used to downregulate RECQ1 expression resulted in similar growth phenotype in HeLa cells. Three independent determinations of cell survival were performed and the mean±SD is presented.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: RECQ1 depletion sensitizes cells to DNA damage induced by ionizing radiation or camptothecin. siRNA knockdown of RECQ1 in HeLa cells leads to increased sensitivity to treatment with IR ( Panel A ) or CPT ( Panel B ). HeLa cells treated with either RECQ1 siRNA or control siRNA (#C) were plated in quadruplicate and treated with increasing doses of IR or concentrations of CPT. Total DNA content was measured as an indication of cell growth. Two independent siRNAs (#L1 and #L2) used to downregulate RECQ1 expression resulted in similar growth phenotype in HeLa cells. Three independent determinations of cell survival were performed and the mean±SD is presented.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Cycling Probe Technology, Expressing

    RECQ1 interacts with Rad51. Panel A, Co-immunoprecipitation of RECQ1 and Rad51 from HeLa nuclear extracts. Rad51 was detected in RECQ1 immunoprecipitates (lane 3). RECQ1 was immmunoprecipitated with Rad51 from nuclear extracts as detected by Western blot (lane 5). Lanes 2 and 4 represent immunoprecipitate from control rabbit and mouse IgG, respectively. Input (lane 1) represent 10% of the total protein used for immunoprecipitation. Panels B and C , ELISA for RECQ1-Rad51 interaction. Either BSA or purified recombinant human RECQ1 (14 nM) was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of purified recombinant human Rad51 (0–76 nM, Panel B or 76 nM, Panel C ) for 1 h at 30°C. In Panel C as indicated, DNaseI (100 U/ml) or ethidium bromide (EtBr) (50 µg/ml) was included in the incubation with Rad51 in the binding step in the corresponding wells to test for DNA-mediated protein interaction. Following washing, RECQ1 bound Rad51 was detected by ELISA using rabbit polyclonal antibody against Rad51. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: RECQ1 interacts with Rad51. Panel A, Co-immunoprecipitation of RECQ1 and Rad51 from HeLa nuclear extracts. Rad51 was detected in RECQ1 immunoprecipitates (lane 3). RECQ1 was immmunoprecipitated with Rad51 from nuclear extracts as detected by Western blot (lane 5). Lanes 2 and 4 represent immunoprecipitate from control rabbit and mouse IgG, respectively. Input (lane 1) represent 10% of the total protein used for immunoprecipitation. Panels B and C , ELISA for RECQ1-Rad51 interaction. Either BSA or purified recombinant human RECQ1 (14 nM) was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of purified recombinant human Rad51 (0–76 nM, Panel B or 76 nM, Panel C ) for 1 h at 30°C. In Panel C as indicated, DNaseI (100 U/ml) or ethidium bromide (EtBr) (50 µg/ml) was included in the incubation with Rad51 in the binding step in the corresponding wells to test for DNA-mediated protein interaction. Following washing, RECQ1 bound Rad51 was detected by ELISA using rabbit polyclonal antibody against Rad51. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Blocking Assay, Incubation, Binding Assay

    RECQ1-depleted cells show reduced cellular proliferation. Panel A , Small interfering RNA inhibition of RECQ1. Whole cell extracts from HeLa cells that had been transfected with control or RECQ1 siRNA (oligonucleotide L1 or L2) were subjected to Western blotting with RECQ1 or Actin antibodies (as a loading control). Panel B , Proliferation of control or RECQ1 siRNA treated cells as determined by CyQuant assay at indicated time points after transfection. Panel C , MTT assay of in vitro proliferation of control or RECQ1-siRNA transfected cells. Panel D , Colorimetric BrdU cell proliferation ELISA of control or RECQ1 siRNA treated HeLa cells. Results are taken from three independent experiments and proliferation is represented as the mean±standard deviation (SD).

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: RECQ1-depleted cells show reduced cellular proliferation. Panel A , Small interfering RNA inhibition of RECQ1. Whole cell extracts from HeLa cells that had been transfected with control or RECQ1 siRNA (oligonucleotide L1 or L2) were subjected to Western blotting with RECQ1 or Actin antibodies (as a loading control). Panel B , Proliferation of control or RECQ1 siRNA treated cells as determined by CyQuant assay at indicated time points after transfection. Panel C , MTT assay of in vitro proliferation of control or RECQ1-siRNA transfected cells. Panel D , Colorimetric BrdU cell proliferation ELISA of control or RECQ1 siRNA treated HeLa cells. Results are taken from three independent experiments and proliferation is represented as the mean±standard deviation (SD).

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Small Interfering RNA, Inhibition, Transfection, Western Blot, CyQUANT Assay, MTT Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Depletion of RECQ1 leads to spontaneous formation of γ-H2AX foci in the absence of exogenous damage. Panel A, Control or RECQ1 siRNA-treated HeLa cells were grown on coverslips, fixed with formaldehyde and co-immunostained with anti- γ-H2AX and anti-RECQ1 antibodies. The merged picture shows cells stained with anti- γ-H2AX (green) and anti-RECQ1 (red) as well as DAPI (blue). Normal induction of γ-H2AX foci is shown upon IR (5 Gy) exposure in RECQ1 depleted cells. Panel B, Quantitative assessment of γ-H2AX foci in control or RECQ1-depleted cells that have been either untreated or exposed to IR, as described in Panel A . Images of at least 100 cells were captured and used for quantitative analyses of γ-H2AX foci. To avoid bias in the selection of cells, DAPI stained nuclei were randomly selected for γ-H2AX staining. Panel C, HeLa cells were treated with control or RECQ1 siRNA. Following IR exposure or not, cell lysates were immunoblotted with anti-RECQ1, anti- γ-H2AX, or anti-actin antibodies.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Depletion of RECQ1 leads to spontaneous formation of γ-H2AX foci in the absence of exogenous damage. Panel A, Control or RECQ1 siRNA-treated HeLa cells were grown on coverslips, fixed with formaldehyde and co-immunostained with anti- γ-H2AX and anti-RECQ1 antibodies. The merged picture shows cells stained with anti- γ-H2AX (green) and anti-RECQ1 (red) as well as DAPI (blue). Normal induction of γ-H2AX foci is shown upon IR (5 Gy) exposure in RECQ1 depleted cells. Panel B, Quantitative assessment of γ-H2AX foci in control or RECQ1-depleted cells that have been either untreated or exposed to IR, as described in Panel A . Images of at least 100 cells were captured and used for quantitative analyses of γ-H2AX foci. To avoid bias in the selection of cells, DAPI stained nuclei were randomly selected for γ-H2AX staining. Panel C, HeLa cells were treated with control or RECQ1 siRNA. Following IR exposure or not, cell lysates were immunoblotted with anti-RECQ1, anti- γ-H2AX, or anti-actin antibodies.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Staining, Selection

    Elevated sister chromatid exchanges in RECQ1-depleted human cells. Panel A, SCEs were assayed in BrdU labeled, giemsa-stained chromosome spreads from control or RECQ1 siRNA treated HeLa cells. A representative spread is shown for spontaneous SCEs in control and RECQ1-depleted cells. Panel B , Quantitative representation of the number of SCEs per metaphase, either spontaneous or induced by CPT or MMC in cells treated with either control or RECQ1 siRNA. A minimum of 25 metaphases were counted for each cell type and treatment.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Elevated sister chromatid exchanges in RECQ1-depleted human cells. Panel A, SCEs were assayed in BrdU labeled, giemsa-stained chromosome spreads from control or RECQ1 siRNA treated HeLa cells. A representative spread is shown for spontaneous SCEs in control and RECQ1-depleted cells. Panel B , Quantitative representation of the number of SCEs per metaphase, either spontaneous or induced by CPT or MMC in cells treated with either control or RECQ1 siRNA. A minimum of 25 metaphases were counted for each cell type and treatment.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Labeling, Staining, Cycling Probe Technology

    Effect of RECQ1 depletion on spontaneous or oxidative stress induced apoptosis. HeLa cells were siRNA treated for 48 h, and then incubated in complete medium for 24 h. Cells were either untreated or incubated with 400 µM H 2 O 2 for 3 h in serum free medium, washed, and allowed to recover in complete medium for 21 h. Panel A, Enrichment of the cytoplasmic histone-associated-DNA fragments that are indicative of an ongoing apoptosis in cells with the indicated siRNA oligonucleotides. Samples were analyzed in duplicates, and data points represent the mean of three independent experiments; bars denote SD. Panel B, immunoblotting analysis of the PARP cleavage in control or RECQ1 siRNA treated HeLa cells either untreated or exposed to 400 µM H 2 O 2 .

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Effect of RECQ1 depletion on spontaneous or oxidative stress induced apoptosis. HeLa cells were siRNA treated for 48 h, and then incubated in complete medium for 24 h. Cells were either untreated or incubated with 400 µM H 2 O 2 for 3 h in serum free medium, washed, and allowed to recover in complete medium for 21 h. Panel A, Enrichment of the cytoplasmic histone-associated-DNA fragments that are indicative of an ongoing apoptosis in cells with the indicated siRNA oligonucleotides. Samples were analyzed in duplicates, and data points represent the mean of three independent experiments; bars denote SD. Panel B, immunoblotting analysis of the PARP cleavage in control or RECQ1 siRNA treated HeLa cells either untreated or exposed to 400 µM H 2 O 2 .

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Incubation

    Phosphorylation of endogenous RECQ1 in response to IR. Panel A, RECQ1 was immunoprecipitated from whole cell extracts of U2OS cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure. The RECQ1 immunoprecipitate from each cell extract was divided in half and was either incubated or not with λ-phosphatase (500 U) for 1 h at 30°C before elution with SDS sample buffer. The immunoprecipitated proteins were resolved on 12% SDS-PAGE, transferred to PVDF membranes, and probed for RECQ1. Panel B, IR-induced RECQ1 phosphorylation is dependent on radiation dose. RECQ1 was immunoprecipitated from either untreated cells or 6 h after treatment of cells with the indicated dose of γ-radiation. Immunoprecipitated proteins were divided in two aliquots which were either treated with λ-phosphatase or left untreated. RECQ1 was detected as described above. Panel C, Time course of RECQ1 phosphorylation in response to IR. U2OS cells were subjected to γ-radiation (10 Gy), and RECQ1 was immunoprecipiated from whole cell extracts prepared at the indicated time points following IR exposure. Panel D, Phosphorylated RECQ1 is preferentially associated with chromatin in IR-treated cells. RECQ1 was immunoprecipitated from the detergent-soluble (S2) and insoluble (S4) fractions of untreated or IR treated cells, resolved on 12% SDS-PAGE, and detected by Western blot analysis.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Phosphorylation of endogenous RECQ1 in response to IR. Panel A, RECQ1 was immunoprecipitated from whole cell extracts of U2OS cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure. The RECQ1 immunoprecipitate from each cell extract was divided in half and was either incubated or not with λ-phosphatase (500 U) for 1 h at 30°C before elution with SDS sample buffer. The immunoprecipitated proteins were resolved on 12% SDS-PAGE, transferred to PVDF membranes, and probed for RECQ1. Panel B, IR-induced RECQ1 phosphorylation is dependent on radiation dose. RECQ1 was immunoprecipitated from either untreated cells or 6 h after treatment of cells with the indicated dose of γ-radiation. Immunoprecipitated proteins were divided in two aliquots which were either treated with λ-phosphatase or left untreated. RECQ1 was detected as described above. Panel C, Time course of RECQ1 phosphorylation in response to IR. U2OS cells were subjected to γ-radiation (10 Gy), and RECQ1 was immunoprecipiated from whole cell extracts prepared at the indicated time points following IR exposure. Panel D, Phosphorylated RECQ1 is preferentially associated with chromatin in IR-treated cells. RECQ1 was immunoprecipitated from the detergent-soluble (S2) and insoluble (S4) fractions of untreated or IR treated cells, resolved on 12% SDS-PAGE, and detected by Western blot analysis.

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot

    Reduced cell growth and aberrant cell cycle progression of RECQ1-depleted cells. Panel A , Short hairpin RNA (shRNA)-mediated RECQ1 depletion in HeLa cells. Western blot showing RECQ1 expression in puromycin resistant HeLa cells transfected with either control or RECQ1 shRNA (#1 or #2). Actin is used as loading control. Proliferation of control or cells transfected with RECQ1-specific shRNA plasmids was determined by Coulter counting the total number of cells at indicated time points ( Panel B ) and by colony forming assay ( Panel C ). Panels D and E , RECQ1 depletion induces G2/M accumulation. Flow cytometry was used to determine cell cycle distribution of control or RECQ1 shRNA transfected cells ( Panel D ) or RECQ1 siRNA transfected cells ( Panel E ).

    Journal: PLoS ONE

    Article Title: Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges

    doi: 10.1371/journal.pone.0001297

    Figure Lengend Snippet: Reduced cell growth and aberrant cell cycle progression of RECQ1-depleted cells. Panel A , Short hairpin RNA (shRNA)-mediated RECQ1 depletion in HeLa cells. Western blot showing RECQ1 expression in puromycin resistant HeLa cells transfected with either control or RECQ1 shRNA (#1 or #2). Actin is used as loading control. Proliferation of control or cells transfected with RECQ1-specific shRNA plasmids was determined by Coulter counting the total number of cells at indicated time points ( Panel B ) and by colony forming assay ( Panel C ). Panels D and E , RECQ1 depletion induces G2/M accumulation. Flow cytometry was used to determine cell cycle distribution of control or RECQ1 shRNA transfected cells ( Panel D ) or RECQ1 siRNA transfected cells ( Panel E ).

    Article Snippet: Although RECQ1 is not implicated as of yet in a human disease, studies of cells from RECQL knockout mice suggested a role of RECQ1 in the maintenance of chromosomal stability and the repair of double strand breaks .

    Techniques: shRNA, Western Blot, Expressing, Transfection, Flow Cytometry, Cytometry

    Hub modules from the PPI network in osteoarthritis patients were constructed. (A) A total of five upregulated proteins constituted a hub PPI module: GOSR1, SEC22C, VAMP7, STX16) and VAMP3. (B and C) Twelve downregulated proteins constituted 2 hub PPI modules: CCL5, GAL, CXCL5, APP, ADCY9, HRH4, CXCR5, RPA1, RAD50, DNA2, RAD17 and RECQL5. PPI, protein-protein interaction. GOSR1, golgi SNAP receptor complex member 1; SEC22C, SEC22 homolog C vesicle trafficking protein; VAMP7, vesicle associated membrane protein 7; STX16, syntaxin 16; VAMP3, vesicle associated membrane protein 3; CCL5, C-C motif chemokine ligand 5; GAL, galanin and GMAP prepropeptide; CXCL5, C-X-C motif chemokine ligand 5; APP, amyloid beta precursor protein; ADCY9, adenylate cyclase 9; HRH4, histamine receptor H4; CXCR5, C-X-C motif chemokine receptor 5; RPA1, replication protein A1; RAD50, RAD50 double strand break repair protein; DNA2, DNA replication helicase/nuclease 2; RAD17, RAD17 checkpoint clamp loader component; RECQL5, RecQ like helicase 5.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Integrated analysis of key mRNAs and lncRNAs in osteoarthritis

    doi: 10.3892/etm.2018.6360

    Figure Lengend Snippet: Hub modules from the PPI network in osteoarthritis patients were constructed. (A) A total of five upregulated proteins constituted a hub PPI module: GOSR1, SEC22C, VAMP7, STX16) and VAMP3. (B and C) Twelve downregulated proteins constituted 2 hub PPI modules: CCL5, GAL, CXCL5, APP, ADCY9, HRH4, CXCR5, RPA1, RAD50, DNA2, RAD17 and RECQL5. PPI, protein-protein interaction. GOSR1, golgi SNAP receptor complex member 1; SEC22C, SEC22 homolog C vesicle trafficking protein; VAMP7, vesicle associated membrane protein 7; STX16, syntaxin 16; VAMP3, vesicle associated membrane protein 3; CCL5, C-C motif chemokine ligand 5; GAL, galanin and GMAP prepropeptide; CXCL5, C-X-C motif chemokine ligand 5; APP, amyloid beta precursor protein; ADCY9, adenylate cyclase 9; HRH4, histamine receptor H4; CXCR5, C-X-C motif chemokine receptor 5; RPA1, replication protein A1; RAD50, RAD50 double strand break repair protein; DNA2, DNA replication helicase/nuclease 2; RAD17, RAD17 checkpoint clamp loader component; RECQL5, RecQ like helicase 5.

    Article Snippet: In the respective PPI networks, a total of 5 up-regulated proteins, including golgi SNAP receptor complex member 1 (GOSR1), SEC22 homolog C vesicle trafficking protein (SEC22C), vesicle associated membrane protein 7 (VAMP7), syntaxin 16 (STX16) and vesicle associated membrane protein 3 (VAMP3; ) and 12 down-regulated proteins including, C-C motif chemokine ligand 5 (CCL5), galanin and GMAP prepropeptide (GAL), C-X-C motif chemokine ligand 5 (CXCL5), amyloid beta precursor protein (APP), adenylate cyclase 9 (ADCY9), histamine receptor H4 (HRH4), C-X-C motif chemokine receptor 5 (CXCR5), replication protein A1 (RPA1), RAD50 double strand break repair protein (RAD50), DNA replication helicase/nuclease 2 (DNA2), RAD17 checkpoint clamp loader component (RAD17) and RecQ like helicase 5 (RECQL5; ) were identified as key proteins/genes.

    Techniques: Construct

    The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Journal: Nucleic Acids Research

    Article Title: Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs

    doi:

    Figure Lengend Snippet: The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Article Snippet: Alternatively, distamycin A and netropsin may induce structural or topological changes to the DNA double helix which impede progression of the WRN and BLM enzymes.

    Techniques: Inhibition, Activity Assay, Incubation, Concentration Assay

    Potent inhibition of WRN and BLM helicase activities on a M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Journal: Nucleic Acids Research

    Article Title: Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs

    doi:

    Figure Lengend Snippet: Potent inhibition of WRN and BLM helicase activities on a M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Article Snippet: Alternatively, distamycin A and netropsin may induce structural or topological changes to the DNA double helix which impede progression of the WRN and BLM enzymes.

    Techniques: Inhibition, Incubation, Activity Assay, Concentration Assay

    Human immature moDCs constantly express NLRC5, NLRX1, RIG-I and MDA5. (A–D) Freshly isolated monocytes were seeded in 24-well plates and differentiated as described in the “Materials and Methods.” The protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were measured by western blot. (A,C) Representative blots are shown. (B,D) Graphs represent the kinetics of protein expressions during moDC differentiation. Data are represented as mean ± SD of 3–5 individual experiments and were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: Human immature moDCs constantly express NLRC5, NLRX1, RIG-I and MDA5. (A–D) Freshly isolated monocytes were seeded in 24-well plates and differentiated as described in the “Materials and Methods.” The protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were measured by western blot. (A,C) Representative blots are shown. (B,D) Graphs represent the kinetics of protein expressions during moDC differentiation. Data are represented as mean ± SD of 3–5 individual experiments and were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Isolation, Western Blot

    NLRX1 but not NLRC5 controls the RIG-I/MDA5 agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–D) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I/MDA5 ligand polyI:C (1 μg/ml). The protein levels of IFN-α, IFN-β (A) , TNF, IL-6, and IL-8 (B) were detected by ELISA after 24 h. (C,D) Kinetics of IκBα degradation was determined by western blotting. (C) A representative blot is shown. (D) Bar graphs show the relative density of IκBα measured at 60 min of stimulation. (A,B,D) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. ** p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: NLRX1 but not NLRC5 controls the RIG-I/MDA5 agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–D) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I/MDA5 ligand polyI:C (1 μg/ml). The protein levels of IFN-α, IFN-β (A) , TNF, IL-6, and IL-8 (B) were detected by ELISA after 24 h. (C,D) Kinetics of IκBα degradation was determined by western blotting. (C) A representative blot is shown. (D) Bar graphs show the relative density of IκBα measured at 60 min of stimulation. (A,B,D) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. ** p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

    NLRX1 but not NLRC5 affects the specific RIG-I agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–E) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml). The mRNA expression levels of IFNA1 and IFNB were assessed by real-time PCR after 12 h (A) and IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C ) protein levels were measured by ELISA after 24 h. (D,E) Kinetics of IκBα degradation was determined by western blotting. (D) A representative blot is shown. (E) Bar graphs show the relative density of IκBα measured at 60 min of stimulation. (A-C, E) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: NLRX1 but not NLRC5 affects the specific RIG-I agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–E) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml). The mRNA expression levels of IFNA1 and IFNB were assessed by real-time PCR after 12 h (A) and IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C ) protein levels were measured by ELISA after 24 h. (D,E) Kinetics of IκBα degradation was determined by western blotting. (D) A representative blot is shown. (E) Bar graphs show the relative density of IκBα measured at 60 min of stimulation. (A-C, E) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Depletion of NLRC5 or NLRX1 enhances the type I IFN production of GEN2.2 cells but does not influence the NF-κB pathway activity in response to VSV infection. (A–E) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h. (A,B) After silencing cells were pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were infected with VSV at the indicated MOIs. The protein levels of IFN-α, IFN-β (A) , TNF, IL-6, and IL-8 (B) were measured by ELISA after 18 h. (C–E) After silencing GEN2.2 cells were exposed to VSV at the indicated MOIs without CpG-A pre-treatment and the protein levels of RIG-I and MDA5 were detected by western blot at 24 h (C) . Concentrations of IFN-α, IFN-β (D) , TNF, IL-6, and IL-8 (E) were measured by ELISA from the supernatant of the VSV-infected cells. (C) A representative blot is shown. Data are represented as means ± SD of 4 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: Depletion of NLRC5 or NLRX1 enhances the type I IFN production of GEN2.2 cells but does not influence the NF-κB pathway activity in response to VSV infection. (A–E) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h. (A,B) After silencing cells were pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were infected with VSV at the indicated MOIs. The protein levels of IFN-α, IFN-β (A) , TNF, IL-6, and IL-8 (B) were measured by ELISA after 18 h. (C–E) After silencing GEN2.2 cells were exposed to VSV at the indicated MOIs without CpG-A pre-treatment and the protein levels of RIG-I and MDA5 were detected by western blot at 24 h (C) . Concentrations of IFN-α, IFN-β (D) , TNF, IL-6, and IL-8 (E) were measured by ELISA from the supernatant of the VSV-infected cells. (C) A representative blot is shown. Data are represented as means ± SD of 4 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Activity Assay, Infection, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    The expression of NLRC5, RIG-I, and MDA5 is inducible in primary human pDCs after CpG-A treatments. (A–D) Freshly isolated primary human pDCs were stimulated with 2.5 μM CpG-A for 16 h, thereafter the protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were detected by western blotting. Representative blots are shown in (A,C) . Data are shown as mean ± SD from 3 to 4 experiments in panels (B,D) . (B,D) Statistical comparisons were performed using Student's t -test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: The expression of NLRC5, RIG-I, and MDA5 is inducible in primary human pDCs after CpG-A treatments. (A–D) Freshly isolated primary human pDCs were stimulated with 2.5 μM CpG-A for 16 h, thereafter the protein levels of NLRC5, NLRX1, RIG-I, MDA5, MAVS, and TBK1 were detected by western blotting. Representative blots are shown in (A,C) . Data are shown as mean ± SD from 3 to 4 experiments in panels (B,D) . (B,D) Statistical comparisons were performed using Student's t -test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Expressing, Isolation, Western Blot

    The specific RIG-I agonist-induced type I IFN production is upregulated by NLRC5 or NLRX1 silencing while the NF-κB signaling pathway is not affected in GEN2.2 cells. (A–D) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h then pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were stimulated with the specific RIG-I agonist 5′ppp-dsRNA (RIGL, 1 μg/ml). The IFNA1 and IFNB mRNA expression levels were assessed by real-time PCR after 3 h (A) and IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C) protein levels were measured by ELISA after 6 (B) or 24 h (C) . (D) Kinetics of IκBα degradation was determined by western blotting. (D) A representative blot is shown. (A–C) Data are represented as means ± SD of 3-5 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: The specific RIG-I agonist-induced type I IFN production is upregulated by NLRC5 or NLRX1 silencing while the NF-κB signaling pathway is not affected in GEN2.2 cells. (A–D) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h then pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were stimulated with the specific RIG-I agonist 5′ppp-dsRNA (RIGL, 1 μg/ml). The IFNA1 and IFNB mRNA expression levels were assessed by real-time PCR after 3 h (A) and IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C) protein levels were measured by ELISA after 6 (B) or 24 h (C) . (D) Kinetics of IκBα degradation was determined by western blotting. (D) A representative blot is shown. (A–C) Data are represented as means ± SD of 3-5 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    The RIG-I/MDA5 agonist-induced type I IFN production is upregulated by NLRC5 or NLRX1 silencing while the NF-κB signaling pathway is not affected in GEN2.2 cells. (A–C) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h then pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were stimulated with the RIG-I/MDA5 agonist polyI:C (1 μg/ml). The protein levels of IFN-α, IFN-β ( A ), TNF, IL-6, and IL-8 (B) were measured by ELISA after 6 (A) or 24 h (B) . (C) Kinetics of IκBα degradation was determined by western blotting. (C) A representative blot is shown. Data are represented as means ± SD of 4 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. ** p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: The RIG-I/MDA5 agonist-induced type I IFN production is upregulated by NLRC5 or NLRX1 silencing while the NF-κB signaling pathway is not affected in GEN2.2 cells. (A–C) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h then pre-treated with 0.25 μM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were stimulated with the RIG-I/MDA5 agonist polyI:C (1 μg/ml). The protein levels of IFN-α, IFN-β ( A ), TNF, IL-6, and IL-8 (B) were measured by ELISA after 6 (A) or 24 h (B) . (C) Kinetics of IκBα degradation was determined by western blotting. (C) A representative blot is shown. Data are represented as means ± SD of 4 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. ** p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    The expression of NLRC5, RIG-I and MDA5 but not that of NLRX1 is upregulated by CpG-A treatment in the human GEN2.2 pDC cell line. (A–E) GEN2.2 cells were treated with increasing concentration of CpG-A (0.25–1 μM) in a time dependent manner. The expression of NLRC5 and NLRX1 was measured at the mRNA level by Q-PCR (A) and at the protein level by western blotting (B,C) . The changes in protein levels of RIG-I, MDA5, MAVS, and TBK1 were also analyzed after CpG-A treatments by western blotting (D,E) . Representative blots are shown in (B,D) . Data are shown as mean ± SD from 4 to 6 independent experiments in panels (A,C,E) . Data were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: The expression of NLRC5, RIG-I and MDA5 but not that of NLRX1 is upregulated by CpG-A treatment in the human GEN2.2 pDC cell line. (A–E) GEN2.2 cells were treated with increasing concentration of CpG-A (0.25–1 μM) in a time dependent manner. The expression of NLRC5 and NLRX1 was measured at the mRNA level by Q-PCR (A) and at the protein level by western blotting (B,C) . The changes in protein levels of RIG-I, MDA5, MAVS, and TBK1 were also analyzed after CpG-A treatments by western blotting (D,E) . Representative blots are shown in (B,D) . Data are shown as mean ± SD from 4 to 6 independent experiments in panels (A,C,E) . Data were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Expressing, Concentration Assay, Polymerase Chain Reaction, Western Blot

    NLRX1 but not NLRC5 affects the type I IFN and pro-inflammatory responses in VSV-infected human moDCs. (A–C) moDCs transfected with the indicated siRNAs were exposed to VSV at the indicated MOIs and after 18 h the protein levels of RIG-I and MDA5 (A) were analyzed by western blotting, and the concentrations of secreted IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C) were determined by ELISA. (A) A representative blot is shown. (B,C) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Journal: Frontiers in Immunology

    Article Title: Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells

    doi: 10.3389/fimmu.2018.02314

    Figure Lengend Snippet: NLRX1 but not NLRC5 affects the type I IFN and pro-inflammatory responses in VSV-infected human moDCs. (A–C) moDCs transfected with the indicated siRNAs were exposed to VSV at the indicated MOIs and after 18 h the protein levels of RIG-I and MDA5 (A) were analyzed by western blotting, and the concentrations of secreted IFN-α, IFN-β (B) , TNF, IL-6, and IL-8 (C) were determined by ELISA. (A) A representative blot is shown. (B,C) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. * p

    Article Snippet: The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778).

    Techniques: Infection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Intercalating agents affect the recruitment of RECQL5 to the sites of ICLs. U2OS cells were transfected with either 2 µg GFP–RECQL5 ( A ) or 2 µg GFP-XPC ( D ). Twenty-four hours posttransfection, the cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( B ) U2OS cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for γ-H2AX (red) to indicate a damage signal. The targeted regions are indicated by arrows. U2OS cells were transfected with either 2 µg GFP-RECQL5 ( C ) or 2 µg GFP-XPC ( E ). Twenty-four hours posttransfection, the cells were incubated with EtBr at 5 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: Intercalating agents affect the recruitment of RECQL5 to the sites of ICLs. U2OS cells were transfected with either 2 µg GFP–RECQL5 ( A ) or 2 µg GFP-XPC ( D ). Twenty-four hours posttransfection, the cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( B ) U2OS cells were incubated with actinomycin-D at 0.05 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for γ-H2AX (red) to indicate a damage signal. The targeted regions are indicated by arrows. U2OS cells were transfected with either 2 µg GFP-RECQL5 ( C ) or 2 µg GFP-XPC ( E ). Twenty-four hours posttransfection, the cells were incubated with EtBr at 5 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Transfection, Incubation, Sequencing

    Topoisomerase poisons and other RecQ helicases do not influence recruitment of RECQL5 to the site of ICLs. U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with either 500 nM camptothecin ( A ) or 6 µM etoposide ( B ) for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( C ) WRN patient fibroblasts (AG11395) and ( D ) BLM patient fibroblasts (GM08505) were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: Topoisomerase poisons and other RecQ helicases do not influence recruitment of RECQL5 to the site of ICLs. U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with either 500 nM camptothecin ( A ) or 6 µM etoposide ( B ) for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( C ) WRN patient fibroblasts (AG11395) and ( D ) BLM patient fibroblasts (GM08505) were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Transfection, Incubation, Sequencing

    RECQL5 facilitates repair of ICLs. ( A ) Representative images from an alkaline comet tail recovery assay of shScrambled and shRECQL5 U2OS cells with and without trioxalen plus UVA treatment and fixed immediately (0h), 6 or 16 h later. Untreated represents the nuclear DNA from cells unexposed to trioxalen. ( B ) Quantitation of the comet tail. Mean tail moment was measured as described in Materials and methods and was normalized relative to 0 h measurement of each sample. More than 100 nuclear DNAs were analyzed in each experiment. Two biologically independent experiments were performed and error bars represent ± SD. *** P

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: RECQL5 facilitates repair of ICLs. ( A ) Representative images from an alkaline comet tail recovery assay of shScrambled and shRECQL5 U2OS cells with and without trioxalen plus UVA treatment and fixed immediately (0h), 6 or 16 h later. Untreated represents the nuclear DNA from cells unexposed to trioxalen. ( B ) Quantitation of the comet tail. Mean tail moment was measured as described in Materials and methods and was normalized relative to 0 h measurement of each sample. More than 100 nuclear DNAs were analyzed in each experiment. Two biologically independent experiments were performed and error bars represent ± SD. *** P

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Quantitation Assay

    WRN, BLM and RECQL5 are recruited to ICLs. ( A ) U2OS cells were transfected with 2 µg of GFP-XPC; 24h posttransfection, the cells were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( B ) U2OS cells were transfected with 2 µg of GFP expressing plasmid and treated as in panel (A). U2OS cells were transfected with 2 µg of ( C ) GFP-BLM, ( D ) GFP-WRN, ( E ) GFP-RECQL5, ( F ) GFP-RECQL1 or ( G ) GFP-RECQL4, and the cells were processed as in panel (A). ( H ) U2OS cells were transfected with 2 µg of GFP-RECQL5 and treated as in panel (A). The targeted regions are indicated by arrows. Bar, 5 µM.

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: WRN, BLM and RECQL5 are recruited to ICLs. ( A ) U2OS cells were transfected with 2 µg of GFP-XPC; 24h posttransfection, the cells were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( B ) U2OS cells were transfected with 2 µg of GFP expressing plasmid and treated as in panel (A). U2OS cells were transfected with 2 µg of ( C ) GFP-BLM, ( D ) GFP-WRN, ( E ) GFP-RECQL5, ( F ) GFP-RECQL1 or ( G ) GFP-RECQL4, and the cells were processed as in panel (A). ( H ) U2OS cells were transfected with 2 µg of GFP-RECQL5 and treated as in panel (A). The targeted regions are indicated by arrows. Bar, 5 µM.

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Transfection, Incubation, Sequencing, Expressing, Plasmid Preparation

    Interaction of RECQL5 deletion mutants with ICLs. ( A ) Schematic illustration of the wild-type and deletion mutants of RECQL5. The helicase, RecQ C-terminal (RQC), KIX and SRI domains are shown along with the nuclear localization signal (NLS) in endogenous RECQL5. The ribbon diagrams below show which GFP-fusion constructs recruit to ICLs. ( B ) Western blot showing the cellular expression of the various deletion mutants. ( C ) U2OS cells were transfected with 2 µg of indicated plasmid; 24h posttransfection, the cells were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: Interaction of RECQL5 deletion mutants with ICLs. ( A ) Schematic illustration of the wild-type and deletion mutants of RECQL5. The helicase, RecQ C-terminal (RQC), KIX and SRI domains are shown along with the nuclear localization signal (NLS) in endogenous RECQL5. The ribbon diagrams below show which GFP-fusion constructs recruit to ICLs. ( B ) Western blot showing the cellular expression of the various deletion mutants. ( C ) U2OS cells were transfected with 2 µg of indicated plasmid; 24h posttransfection, the cells were incubated with 6 µM trioxalen for 20 min and then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Construct, Western Blot, Expressing, Transfection, Plasmid Preparation, Incubation, Sequencing

    Inhibition of transcription does not perturb recruitment of RECQL5 to ICLs. ( A ) U2OS cells were transfected with the indicated plasmids. After 24h, the cells were treated with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for FLAG (green) to detect the KIX-Flag protein and endogenous XPC (red) to indicate a damage signal. The targeted regions are indicated by the arrows. ( B ) Western blot showing the cellular expression of the KIX domain and the E584D KIX constructs. ( C ) U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with α-amanitin at 50 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( D ) U2OS cells were incubated with α-amanitin at 50 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for endogenous RECQL5 (green) and for the γ-H2AX (red) for damage signal. ( E ) U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with DRB at 80 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Journal: Carcinogenesis

    Article Title: The RecQ helicase RECQL5 participates in psoralen-induced interstrand cross-link repair

    doi: 10.1093/carcin/bgt183

    Figure Lengend Snippet: Inhibition of transcription does not perturb recruitment of RECQL5 to ICLs. ( A ) U2OS cells were transfected with the indicated plasmids. After 24h, the cells were treated with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for FLAG (green) to detect the KIX-Flag protein and endogenous XPC (red) to indicate a damage signal. The targeted regions are indicated by the arrows. ( B ) Western blot showing the cellular expression of the KIX domain and the E584D KIX constructs. ( C ) U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with α-amanitin at 50 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. ( D ) U2OS cells were incubated with α-amanitin at 50 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nucleus in individual cells, fixed after 5 min and immunostained for endogenous RECQL5 (green) and for the γ-H2AX (red) for damage signal. ( E ) U2OS cells were transfected with 2 µg GFP-RECQL5. Twenty-four hours posttransfection, the cells were incubated with DRB at 80 µg/ml for 2 h and with 6 µM trioxalen for 20 min, then laser photoactivated in the nuclear region in individual cells in a defined time sequence. The targeted regions are indicated by arrows. Bar, 5 µm.

    Article Snippet: Biochemical studies suggest that RECQL5 may bind DNA in a structure-specific manner not in a sequence-specific manner ( , ).

    Techniques: Inhibition, Transfection, Western Blot, Expressing, Construct, Incubation, Sequencing

    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: Ligation, Incubation, Marker, In Vitro, Non-Homologous End Joining, Purification, Staining, Agarose Gel Electrophoresis, Western Blot

    A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: In Vitro, Incubation, SDS Page, Western Blot, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Binding Assay, Staining, Marker, Molecular Weight

    RECQ1 and Ku70/80 co-bind a linear blunt duplex DNA. A. RECQ1 binds a 322 bp blunt duplex fragment derived from pUC19 plasmid DNA. An electrophoretic mobility shift assay (EMSA) was performed to examine the ability of increasing concentration of purified RECQ1 to bind linearized plasmid DNA (30 ng) under DNA binding conditions as described in materials and methods. Protein-DNA complexes were resolved by native 6% polyacrylamide gels and detected by staining with SYBR Gold and a typical inverted image is shown. B. RECQ1 facilitates the formation of higher order DNA complex with Ku70/80. EMSA was performed to examine the ability of increasing concentration of Ku70/80 (0–100 nM) to interact with RECQ1 (12.5 nM) when bound to linear plasmid DNA (30 ng). Both RECQ1 and Ku70/80 bind to blunt duplex resulting in a series of progressively retarded bands, but the mobility of RECQ1-DNA and Ku70/80-DNA complex is distinct. Additional slow migrating bands were observed in Ku70/80 (25, 50 and 100 nM) in the presence of RECQ1 (12.5 nM) (lanes 4–6 and 7–9). C. Molar excess of RECQ1 may compete with Ku70/80 for DNA binding. EMSA was performed to examine the ability of increasing concentration of RECQ1 (0–100 nM) to interact with Ku70/80 (12.5 nM) when bound to linearized plasmid DNA (30 ng) (lane 7 and 8). Arrow indicates the change in band shift pattern of DNA-protein complexes at 50 nM RECQ1. D. RECQ1 and Ku70/80 are co-bound to linear blunt duplex. EMSA reactions were performed using biotinylated DNA probe and the indicated concentration of RECQ1, Ku70/80 or both. The DNA probe was either exposed to a mixture of RECQ1 and Ku70/80, or pre-incubated with one protein followed by addition of the second protein as indicated by parentheses. The DNA-protein complexes were pulled-down on streptavidin magnetic beads and DNA-bound RECQ1 and Ku70/80 were analyzed by Western blotting. Comparable amount of DNA was pull-down in all reactions as shown by agarose gel analyses (bottom). Lane 1 represents biotinylated DNA bound to the streptavidin beads in the absence of RECQ1 or Ku70/80. DNA size marker is shown in leftmost lane of each gel.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1 and Ku70/80 co-bind a linear blunt duplex DNA. A. RECQ1 binds a 322 bp blunt duplex fragment derived from pUC19 plasmid DNA. An electrophoretic mobility shift assay (EMSA) was performed to examine the ability of increasing concentration of purified RECQ1 to bind linearized plasmid DNA (30 ng) under DNA binding conditions as described in materials and methods. Protein-DNA complexes were resolved by native 6% polyacrylamide gels and detected by staining with SYBR Gold and a typical inverted image is shown. B. RECQ1 facilitates the formation of higher order DNA complex with Ku70/80. EMSA was performed to examine the ability of increasing concentration of Ku70/80 (0–100 nM) to interact with RECQ1 (12.5 nM) when bound to linear plasmid DNA (30 ng). Both RECQ1 and Ku70/80 bind to blunt duplex resulting in a series of progressively retarded bands, but the mobility of RECQ1-DNA and Ku70/80-DNA complex is distinct. Additional slow migrating bands were observed in Ku70/80 (25, 50 and 100 nM) in the presence of RECQ1 (12.5 nM) (lanes 4–6 and 7–9). C. Molar excess of RECQ1 may compete with Ku70/80 for DNA binding. EMSA was performed to examine the ability of increasing concentration of RECQ1 (0–100 nM) to interact with Ku70/80 (12.5 nM) when bound to linearized plasmid DNA (30 ng) (lane 7 and 8). Arrow indicates the change in band shift pattern of DNA-protein complexes at 50 nM RECQ1. D. RECQ1 and Ku70/80 are co-bound to linear blunt duplex. EMSA reactions were performed using biotinylated DNA probe and the indicated concentration of RECQ1, Ku70/80 or both. The DNA probe was either exposed to a mixture of RECQ1 and Ku70/80, or pre-incubated with one protein followed by addition of the second protein as indicated by parentheses. The DNA-protein complexes were pulled-down on streptavidin magnetic beads and DNA-bound RECQ1 and Ku70/80 were analyzed by Western blotting. Comparable amount of DNA was pull-down in all reactions as shown by agarose gel analyses (bottom). Lane 1 represents biotinylated DNA bound to the streptavidin beads in the absence of RECQ1 or Ku70/80. DNA size marker is shown in leftmost lane of each gel.

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: Derivative Assay, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Concentration Assay, Purification, Binding Assay, Staining, Incubation, Magnetic Beads, Western Blot, Agarose Gel Electrophoresis, Marker

    RECQ1 interacts with Ku70/80 in vivo . A . Co-immunoprecipitation analysis of RECQ1 interaction with Ku70/80 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, Ku70/80 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained Ku70 and Ku80 subunits but DNA-PKcs was not detected. Reciprocal co-IPs of Ku70/80 also contained RECQ1. B. Association of RECQ1 and Ku70/80 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and Ku70/80 using benzonase-treated extract in IP reaction. C. RECQ1 interacts with Ku in DNA-PKcs deficient and proficient cells. Lysates of MO59J (DNA-PKcs deficient) or MO59K (DNA-PKcs proficient) cells were used for IP using RECQ1 antibody or IgG and analyzed by Western blotting as indicated. D . Immunofluorescence staining of endogenous RECQ1 and Ku70/80. HeLa cells grown on coverslips were either mock-treated or treated with NCS (100 ng/ml, 3 h). Cells were fixed and immunostained using a mouse monoclonal Ku70/80 antibody (1∶200) and a rabbit polyclonal RECQ1 antibody (1∶500). RECQ1 and Ku70/80 were visualized with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies, respectively, followed by confocal microscopy. Inset shows enlarged portion of the nucleus after NCS treatment; colocalization of RECQ1 (green) and Ku70/80 (red) in cells appears yellow in merged images. In all experiments, input corresponds to 5% of total protein used in IP reactions.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1 interacts with Ku70/80 in vivo . A . Co-immunoprecipitation analysis of RECQ1 interaction with Ku70/80 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, Ku70/80 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained Ku70 and Ku80 subunits but DNA-PKcs was not detected. Reciprocal co-IPs of Ku70/80 also contained RECQ1. B. Association of RECQ1 and Ku70/80 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and Ku70/80 using benzonase-treated extract in IP reaction. C. RECQ1 interacts with Ku in DNA-PKcs deficient and proficient cells. Lysates of MO59J (DNA-PKcs deficient) or MO59K (DNA-PKcs proficient) cells were used for IP using RECQ1 antibody or IgG and analyzed by Western blotting as indicated. D . Immunofluorescence staining of endogenous RECQ1 and Ku70/80. HeLa cells grown on coverslips were either mock-treated or treated with NCS (100 ng/ml, 3 h). Cells were fixed and immunostained using a mouse monoclonal Ku70/80 antibody (1∶200) and a rabbit polyclonal RECQ1 antibody (1∶500). RECQ1 and Ku70/80 were visualized with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies, respectively, followed by confocal microscopy. Inset shows enlarged portion of the nucleus after NCS treatment; colocalization of RECQ1 (green) and Ku70/80 (red) in cells appears yellow in merged images. In all experiments, input corresponds to 5% of total protein used in IP reactions.

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: In Vivo, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

    RECQ1-null cells promote end-joining but show reduced Ku-DNA binding activity. A. Level of in vitro end-joining activity is comparable in WT and RECQ1 KO MEFs. Cell free extracts prepared from non-transformed RECQ1 WT or KO MEFs were used in end-joining reaction containing EcoRI-linearized pUC19 DNA as substrate. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer, trimer and tetramer are indicated (right panel). Western blot showed no detectable difference in Ku protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. B. Presence of PARP-1 antibody interferes with RECQ1 KO cell free extract mediated end-joining. In addition to standard reactions, in vitro end-joining reactions were performed with WT or KO cell extracts in the presence of a DNA-PKcs inhibitor Nu7026 (1.2 µM) or a specific anti-PARP-1 antibody (3 µg). IgG (3 µg) was included as unrelated antibody in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Western blot showed no detectable difference in PARP-1 protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. C. Ku70/80 DNA binding assay performed by using Active Motif kit shows diminished DNA binding in RECQ1 KO extract as compared to WT extract (p

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1-null cells promote end-joining but show reduced Ku-DNA binding activity. A. Level of in vitro end-joining activity is comparable in WT and RECQ1 KO MEFs. Cell free extracts prepared from non-transformed RECQ1 WT or KO MEFs were used in end-joining reaction containing EcoRI-linearized pUC19 DNA as substrate. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer, trimer and tetramer are indicated (right panel). Western blot showed no detectable difference in Ku protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. B. Presence of PARP-1 antibody interferes with RECQ1 KO cell free extract mediated end-joining. In addition to standard reactions, in vitro end-joining reactions were performed with WT or KO cell extracts in the presence of a DNA-PKcs inhibitor Nu7026 (1.2 µM) or a specific anti-PARP-1 antibody (3 µg). IgG (3 µg) was included as unrelated antibody in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Western blot showed no detectable difference in PARP-1 protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. C. Ku70/80 DNA binding assay performed by using Active Motif kit shows diminished DNA binding in RECQ1 KO extract as compared to WT extract (p

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: Binding Assay, Activity Assay, In Vitro, Transformation Assay, Western Blot, DNA Binding Assay

    RECQ1 helicase binds and unwinds a Ku-bound forked duplex DNA substrate. A. RECQ1 binds and unwinds Ku-bound fork duplex. Ku70/80 (12.5 nM), RECQ1 (17.3 nM), or both were incubated with radiolabeled fork duplex (0.5 nM) in DNA binding buffer containing ATPγS or ATP (2 mm) as described in materials and methods. The protein-DNA complexes were then resolved on native 5% polyacrylamide gels. Radiolabeled bands were detected by PhosphorImager and a typical gel of resolved RECQ1/Ku70/80-fork duplex mixture is shown. Ku-DNA, RECQ1-DNA and RECQ1-Ku-DNA complexes are indicated based on their gel mobility shift. When binding reaction mixtures contained ATP, fork duplex, either alone or in complex with Ku70/80, was unwound by RECQ1 helicase resulting in the appearance of a faster migrating single stranded oligonucleotide as indicated. B. Quantitative comparison of RECQ1 unwinding of fork duplex or a Ku70/80-bound fork duplex assayed under DNA binding conditions in the presence of ATP (2 mM). The results shown are the average of at least three independent experiments, with SD indicated by error bars. C. Intrinsic ATPase activity of RECQ1 is essential for unwinding of Ku-bound fork duplex. Unwinding of Ku-bound fork duplex mediated by the RECQ1 K119A , RECQ1 K119R or wild-type RECQ1 WT (each 17.3 nM) was assayed under DNA-binding conditions in the presence of ATP (2 mm). D. RECQ1 helicase activity is inhibited at several fold molar excess of Ku70/80. Helicase reactions containing fork duplex DNA substrate (0.5 nM) and the specified concentrations of Ku70/80 in the presence or absence of RECQ1 (2 nM) were incubated at 37°C for 15 min under standard helicase assay conditions as described in materials and methods. Phosphorimage of a typical gel is shown. Δ, heat-denatured DNA substrate control.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1 helicase binds and unwinds a Ku-bound forked duplex DNA substrate. A. RECQ1 binds and unwinds Ku-bound fork duplex. Ku70/80 (12.5 nM), RECQ1 (17.3 nM), or both were incubated with radiolabeled fork duplex (0.5 nM) in DNA binding buffer containing ATPγS or ATP (2 mm) as described in materials and methods. The protein-DNA complexes were then resolved on native 5% polyacrylamide gels. Radiolabeled bands were detected by PhosphorImager and a typical gel of resolved RECQ1/Ku70/80-fork duplex mixture is shown. Ku-DNA, RECQ1-DNA and RECQ1-Ku-DNA complexes are indicated based on their gel mobility shift. When binding reaction mixtures contained ATP, fork duplex, either alone or in complex with Ku70/80, was unwound by RECQ1 helicase resulting in the appearance of a faster migrating single stranded oligonucleotide as indicated. B. Quantitative comparison of RECQ1 unwinding of fork duplex or a Ku70/80-bound fork duplex assayed under DNA binding conditions in the presence of ATP (2 mM). The results shown are the average of at least three independent experiments, with SD indicated by error bars. C. Intrinsic ATPase activity of RECQ1 is essential for unwinding of Ku-bound fork duplex. Unwinding of Ku-bound fork duplex mediated by the RECQ1 K119A , RECQ1 K119R or wild-type RECQ1 WT (each 17.3 nM) was assayed under DNA-binding conditions in the presence of ATP (2 mm). D. RECQ1 helicase activity is inhibited at several fold molar excess of Ku70/80. Helicase reactions containing fork duplex DNA substrate (0.5 nM) and the specified concentrations of Ku70/80 in the presence or absence of RECQ1 (2 nM) were incubated at 37°C for 15 min under standard helicase assay conditions as described in materials and methods. Phosphorimage of a typical gel is shown. Δ, heat-denatured DNA substrate control.

    Article Snippet: Collectively, these results demonstrate a physical and functional interaction between RECQ1 and Ku70/80, and suggest a role of RECQ1 in DSB repair via NHEJ.

    Techniques: Incubation, Binding Assay, Mobility Shift, Activity Assay, Helicase Assay

    Transfection with CHD7 dominant-negative (DN) mRNA transcripts disrupted differentiation potential and cell proliferation of ESCs. ( A ) Protocol for transfection with CHD7 ). Transcripts were transfected into KhES-1 cells on day 0, and the cells were then transferred to low-attachment plates for 24 h, followed by culture in Es6 medium with Rock Inhibitor (RI) for 24 h for EB formation. Microscopic observation of EBs and gene expression profiles by qRT-PCR scorecard panel on day 3 after transfection with DN mRNA. ( B ) CHD7 DN 1 and CHD7 DN2 expression levels were determined by qRT-PCR. A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of day 3-EBs from non-transfected, CHD7 DN1-, CHD7 DN2-, CHD7 -DN1 + CHD7 -DN2-transfected, and mock mRNA-transfected KhES-1 3 days after transfection. Gene expression profiles were determined using qRT-PCR scorecard panels and are shown below the relevant photograph. The cell number on day 3 of culture in one well of a 6-well plate was scored and appended at the top right corner of the relevant image. Non-transfected KhES-1 cultured with Es8 on day 0 (left panel) was used as a control. Representative results of 3 independent experiments are shown. Scale bar: 1 mm.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: Transfection with CHD7 dominant-negative (DN) mRNA transcripts disrupted differentiation potential and cell proliferation of ESCs. ( A ) Protocol for transfection with CHD7 ). Transcripts were transfected into KhES-1 cells on day 0, and the cells were then transferred to low-attachment plates for 24 h, followed by culture in Es6 medium with Rock Inhibitor (RI) for 24 h for EB formation. Microscopic observation of EBs and gene expression profiles by qRT-PCR scorecard panel on day 3 after transfection with DN mRNA. ( B ) CHD7 DN 1 and CHD7 DN2 expression levels were determined by qRT-PCR. A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of day 3-EBs from non-transfected, CHD7 DN1-, CHD7 DN2-, CHD7 -DN1 + CHD7 -DN2-transfected, and mock mRNA-transfected KhES-1 3 days after transfection. Gene expression profiles were determined using qRT-PCR scorecard panels and are shown below the relevant photograph. The cell number on day 3 of culture in one well of a 6-well plate was scored and appended at the top right corner of the relevant image. Non-transfected KhES-1 cultured with Es8 on day 0 (left panel) was used as a control. Representative results of 3 independent experiments are shown. Scale bar: 1 mm.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Transfection, Dominant Negative Mutation, Expressing, Quantitative RT-PCR, Cell Culture

    Downregulation of CHD7 disrupted differentiation in EB formation assays. ( A ) Protocol for EB formation and siCHD7 transfection. Cells were transfected with small double-stranded interfering RNA targeting CHD7 ( siCHD7 ) or nonspecific control siRNA (mock) on day 0. Cells were transferred to low-attachment plates 24 h after transfection with siRNA and cultured with Es6 supplemented with ROCK inhibitor (RI) for 24 h. The medium was then exchanged with fresh Es6 medium and cultured for another 72 h. The morphology of EBs and their gene expression profiles on days 4, 5 and 14 after transfection with siRNA were determined using a qRT-PCR scorecard panel. ( B ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells determined by qRT-PCR (time course sampling). Gene expression of CHD7 was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium, which was independently measured 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) by Western blotting (sampling at day 2). ( D ) Photographs of non-transfected EBs (upper panels) and siCHD7 -transfected (middle panels) or control siRNA-transfected (mock, lower panels) KhES-1 cells on days 4, 5, and 14 are shown. Gene expression profiles of KhES-1 cells under the designated conditions, as determined by qRT-PCR scorecard panel, are shown below the relevant photographs. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: Downregulation of CHD7 disrupted differentiation in EB formation assays. ( A ) Protocol for EB formation and siCHD7 transfection. Cells were transfected with small double-stranded interfering RNA targeting CHD7 ( siCHD7 ) or nonspecific control siRNA (mock) on day 0. Cells were transferred to low-attachment plates 24 h after transfection with siRNA and cultured with Es6 supplemented with ROCK inhibitor (RI) for 24 h. The medium was then exchanged with fresh Es6 medium and cultured for another 72 h. The morphology of EBs and their gene expression profiles on days 4, 5 and 14 after transfection with siRNA were determined using a qRT-PCR scorecard panel. ( B ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells determined by qRT-PCR (time course sampling). Gene expression of CHD7 was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium, which was independently measured 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) by Western blotting (sampling at day 2). ( D ) Photographs of non-transfected EBs (upper panels) and siCHD7 -transfected (middle panels) or control siRNA-transfected (mock, lower panels) KhES-1 cells on days 4, 5, and 14 are shown. Gene expression profiles of KhES-1 cells under the designated conditions, as determined by qRT-PCR scorecard panel, are shown below the relevant photographs. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Sampling, Western Blot

    CHD7 levels mediated the differentiation potential of PSCs. ( A ) The copy numbers of CHD7 isoform 1 in PSCs (ESC: H9, KhES-1, iPSC: PFX#9, 201B7, SHh#2) and their passage numbers (P) are listed in the table. Cells were culture either on feeder with iPSC medium in cell clumps or with Es8, S-P, S-P ACF or RFF2 on VNT-N in single cells or in cell clumps (S-P ACF) ND: Not Determined. Cells cultured with RFF2 on VNT-N failed to demonstrate differentiation potential in EB formation assay (gray columns). ( B ) PSCs (ESC:H9, KhES-1, iPSC:PFX#9, 201B7, SHh#2) were cultured either on feeder with iPSC medium in cell clumps (on feeder cell clumps) or on VNT-N with Es8 in single cells (Es8/N single cells) or S-P ACF in cell clumps (S-P ACF/N clumps) or in single cells (S-P ACF/N single cells), and their differentiation potential was examined by the gene expression profiles of day 14-EBs derived them. The copy numbers of CHD7 prior to EB formation assay was determined and appended beneath the relevant graph.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: CHD7 levels mediated the differentiation potential of PSCs. ( A ) The copy numbers of CHD7 isoform 1 in PSCs (ESC: H9, KhES-1, iPSC: PFX#9, 201B7, SHh#2) and their passage numbers (P) are listed in the table. Cells were culture either on feeder with iPSC medium in cell clumps or with Es8, S-P, S-P ACF or RFF2 on VNT-N in single cells or in cell clumps (S-P ACF) ND: Not Determined. Cells cultured with RFF2 on VNT-N failed to demonstrate differentiation potential in EB formation assay (gray columns). ( B ) PSCs (ESC:H9, KhES-1, iPSC:PFX#9, 201B7, SHh#2) were cultured either on feeder with iPSC medium in cell clumps (on feeder cell clumps) or on VNT-N with Es8 in single cells (Es8/N single cells) or S-P ACF in cell clumps (S-P ACF/N clumps) or in single cells (S-P ACF/N single cells), and their differentiation potential was examined by the gene expression profiles of day 14-EBs derived them. The copy numbers of CHD7 prior to EB formation assay was determined and appended beneath the relevant graph.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Cell Culture, Tube Formation Assay, Expressing, Derivative Assay

    Upregulafx1tion of CHD7 isoform 2 mRNA induced “spontaneous” differentiation in ESCs. ( A ) Protocol for cell culture and transfection with mCHD7 . mCHD7 or control mRNA (mock) was transfected into KhES-1 cells on day 0 and day 1 (two times). Cells were passaged on day 2, reseeded at 3 × 10 5 cells/well in 6-well plates, and cultured another 24 h. ( B ) Expression of CHD7 after mCHD7 or control mRNA ( GFP -transfected; mock) transfection (day 0) was determined by qRT-PCR (time course sampling). CHD7 gene expression by qRT-PCR was standardized according to the average CHD7 expression in KhES-1 cells cultured with RFF2 medium measured independently 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Western blotting (sampling at day 3) of CHD7 in KhES-1 after mCHD7 or control mRNA ( GFP -transfected; mock) was transfected at day 0. NT: non-transfected control. ( D ) The growth of mock-transfected KhES-1 cells (green line) was comparable to that in non-transfected control cells (blue line), whereas that of mCHD7 -transfected KhES-1 cells (red line) was dramatically suppressed on days 2 and 3. ( E ) Photographs of non-transfected (upper panels), mCHD7 -transfected (middle panels), and mock mRNA-transfected (lower panels) KhES-1 cells cultured with RFF2 medium on days 1, 2 and 3. The gene expression profiles obtained by qRT-PCR scorecard panel are presented below the relevant photo. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: Upregulafx1tion of CHD7 isoform 2 mRNA induced “spontaneous” differentiation in ESCs. ( A ) Protocol for cell culture and transfection with mCHD7 . mCHD7 or control mRNA (mock) was transfected into KhES-1 cells on day 0 and day 1 (two times). Cells were passaged on day 2, reseeded at 3 × 10 5 cells/well in 6-well plates, and cultured another 24 h. ( B ) Expression of CHD7 after mCHD7 or control mRNA ( GFP -transfected; mock) transfection (day 0) was determined by qRT-PCR (time course sampling). CHD7 gene expression by qRT-PCR was standardized according to the average CHD7 expression in KhES-1 cells cultured with RFF2 medium measured independently 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Western blotting (sampling at day 3) of CHD7 in KhES-1 after mCHD7 or control mRNA ( GFP -transfected; mock) was transfected at day 0. NT: non-transfected control. ( D ) The growth of mock-transfected KhES-1 cells (green line) was comparable to that in non-transfected control cells (blue line), whereas that of mCHD7 -transfected KhES-1 cells (red line) was dramatically suppressed on days 2 and 3. ( E ) Photographs of non-transfected (upper panels), mCHD7 -transfected (middle panels), and mock mRNA-transfected (lower panels) KhES-1 cells cultured with RFF2 medium on days 1, 2 and 3. The gene expression profiles obtained by qRT-PCR scorecard panel are presented below the relevant photo. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Cell Culture, Transfection, Expressing, Quantitative RT-PCR, Sampling, Western Blot

    Downregulation of CHD7 disrupted the proliferation of ESCs cultured with Es8 medium. ( A ) Protocol for siCHD7 transfection. siCHD7 or nonspecific control siRNA (mock) was transfected into KhES-1 cells on day 0 and day 1. Medium was changed (CM) every day. On days 0–3, cells were harvested for cell counting, and CHD7 expression was determined by qRT-PCR. ( B ) CHD7 gene expression in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells was determined by qRT-PCR. CHD7 gene expression was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium (independently measured 3 times). A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of KhES-1 cells without transfection (upper panels) or with siCHD7 (middle panels) or control siRNA transfection (mock, lower panels) on day 3. The cell number scored is appended at the upper right corner of the respective photograph. ( D ) The cell number was scored for the indicated conditions at the designated day.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: Downregulation of CHD7 disrupted the proliferation of ESCs cultured with Es8 medium. ( A ) Protocol for siCHD7 transfection. siCHD7 or nonspecific control siRNA (mock) was transfected into KhES-1 cells on day 0 and day 1. Medium was changed (CM) every day. On days 0–3, cells were harvested for cell counting, and CHD7 expression was determined by qRT-PCR. ( B ) CHD7 gene expression in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells was determined by qRT-PCR. CHD7 gene expression was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium (independently measured 3 times). A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of KhES-1 cells without transfection (upper panels) or with siCHD7 (middle panels) or control siRNA transfection (mock, lower panels) on day 3. The cell number scored is appended at the upper right corner of the respective photograph. ( D ) The cell number was scored for the indicated conditions at the designated day.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Cell Culture, Transfection, Cell Counting, Expressing, Quantitative RT-PCR

    The differentiation potential of ESCs was altered by culture conditions. ( A ) KhES-1 ESCs in single-cell suspensions were seeded on VNT-N-coated dishes and cultured with Essential 8 (Es8) for 5 passages. The cells were then collected for embryoid body (EB) formation or transferred to Repro FF2 culture medium (RFF2). KhES-1 cells were cultured for 5 passages and collected for EB formation or transferred to Es8 again. KhES-1 cells were cultured for 5 passages, followed by EB formation assays. Photographs of KhES-1 cultures with Es8 or RFF2 medium (upper) at day 1 of culture; EBs at day 14 (lower) are shown. Gene expression profiles of cells in the indicated culture conditions were determined by a qRT-PCR scorecard panel and appended below the relevant photograph. Scale bar: 1.0 mm. ( B ) List of candidate genes and short description related to differentiation potential of PSCs. Value of methylation status and corresponding its gene expression in PSCs cultured with RFF2, S-P and Es8 and shown as in the order of RFF2/S-P/Es8. ( C ) Schematics of CHD7 isoforms, location of PCR primers and antibody (upper), and CHD7 mRNA transcripts used in this study (lower). ( D ) Gene expression of CHD7 in KhES-1 cultures with Es8 (P5 and P15) or RFF2 medium (P5 and P15) determined by qRT-PCR. P: passage numbers. (n = 3 analytical replicates). ( E ) Copy numbers of CHD7 isoform 1 or isoform X4 determined by digital-PCR. Five ng of total RNA obtained from KhES-1 cells cultured with Es8 or RFF2 medium were used as the template, and the copy numbers of the isoforms in each RNA sample were calculated using primer set listed in Fig. 1C. ( F ) CHD7 isoforms detection by Western blotting. Total cell lysates (5.3 μg) from KhES-1 cells cultured with Es8 (P11) or RFF2 medium (P21) were applied to the indicated lanes. CHD7 isoform 1 (expected mass 336 kDa), isoform 2 (101 kDa), and isoform X4 (183 kDa) were detected with antibodies for human CHD7. The signal was visualized by a secondary antibody linked to horseradish peroxidase.

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: The differentiation potential of ESCs was altered by culture conditions. ( A ) KhES-1 ESCs in single-cell suspensions were seeded on VNT-N-coated dishes and cultured with Essential 8 (Es8) for 5 passages. The cells were then collected for embryoid body (EB) formation or transferred to Repro FF2 culture medium (RFF2). KhES-1 cells were cultured for 5 passages and collected for EB formation or transferred to Es8 again. KhES-1 cells were cultured for 5 passages, followed by EB formation assays. Photographs of KhES-1 cultures with Es8 or RFF2 medium (upper) at day 1 of culture; EBs at day 14 (lower) are shown. Gene expression profiles of cells in the indicated culture conditions were determined by a qRT-PCR scorecard panel and appended below the relevant photograph. Scale bar: 1.0 mm. ( B ) List of candidate genes and short description related to differentiation potential of PSCs. Value of methylation status and corresponding its gene expression in PSCs cultured with RFF2, S-P and Es8 and shown as in the order of RFF2/S-P/Es8. ( C ) Schematics of CHD7 isoforms, location of PCR primers and antibody (upper), and CHD7 mRNA transcripts used in this study (lower). ( D ) Gene expression of CHD7 in KhES-1 cultures with Es8 (P5 and P15) or RFF2 medium (P5 and P15) determined by qRT-PCR. P: passage numbers. (n = 3 analytical replicates). ( E ) Copy numbers of CHD7 isoform 1 or isoform X4 determined by digital-PCR. Five ng of total RNA obtained from KhES-1 cells cultured with Es8 or RFF2 medium were used as the template, and the copy numbers of the isoforms in each RNA sample were calculated using primer set listed in Fig. 1C. ( F ) CHD7 isoforms detection by Western blotting. Total cell lysates (5.3 μg) from KhES-1 cells cultured with Es8 (P11) or RFF2 medium (P21) were applied to the indicated lanes. CHD7 isoform 1 (expected mass 336 kDa), isoform 2 (101 kDa), and isoform X4 (183 kDa) were detected with antibodies for human CHD7. The signal was visualized by a secondary antibody linked to horseradish peroxidase.

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Methylation, Polymerase Chain Reaction, Digital PCR, Western Blot

    Human CHD7 co-localized with suppressive chromatin remodelers. ( A ) The binding sites for 17 factors in the map were obtained from a public ESC H1 ChIP-seq database. The odds ratio representing the correlation between binding sites for each pair of factors was calculated. Green indicates high homology between factors, and red indicates no high homology between factors. ( B ) ChIP-qPCR with anti-CHD7 antibody for mock - or si CHD7 -transfected H9 cells. The mock - or si CHD7 -transfected H9 cells in undifferentiated state were immune-precipitated (IP) either with anti-CHD7 antibody or control IgG. qPCR for the putative promoter region of POU5F1 , NANOG , EP300 , EZH2 , SUZ12 or BRG1 were conducted for IP samples. Respective bar shows as fold enrichment of quantity of amplified target gene of interest by IP with anti-CHD7 antibody against that with control IgG in mock - or si CHD7 H9 transfectants. (n = 3 analytical replicates).

    Journal: Scientific Reports

    Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

    doi: 10.1038/s41598-017-18439-y

    Figure Lengend Snippet: Human CHD7 co-localized with suppressive chromatin remodelers. ( A ) The binding sites for 17 factors in the map were obtained from a public ESC H1 ChIP-seq database. The odds ratio representing the correlation between binding sites for each pair of factors was calculated. Green indicates high homology between factors, and red indicates no high homology between factors. ( B ) ChIP-qPCR with anti-CHD7 antibody for mock - or si CHD7 -transfected H9 cells. The mock - or si CHD7 -transfected H9 cells in undifferentiated state were immune-precipitated (IP) either with anti-CHD7 antibody or control IgG. qPCR for the putative promoter region of POU5F1 , NANOG , EP300 , EZH2 , SUZ12 or BRG1 were conducted for IP samples. Respective bar shows as fold enrichment of quantity of amplified target gene of interest by IP with anti-CHD7 antibody against that with control IgG in mock - or si CHD7 H9 transfectants. (n = 3 analytical replicates).

    Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Amplification