Journal: Nucleic Acids Research

Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

doi: 10.1093/nar/gkw730

Figure Lengend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

Article Snippet: TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

Journal: Nucleic Acids Research

Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

doi: 10.1093/nar/gkw730

Figure Lengend Snippet: ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

Article Snippet: TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ).

Techniques: Staining, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Expressing

Journal: PLoS ONE

Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

doi: 10.1371/journal.pone.0052584

Figure Lengend Snippet: A comparison of the methods based on HCl, DNase I and copper(I) ions. A ) The detection of PCNA in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – PCNA) is shown. All of the images have been acquired at 83 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – PCNA image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm. B ) The detection of Cdc45 protein in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – Cdc45) is shown. All of the images have been acquired at 170 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – Cdc45 image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm.

Article Snippet: Alternative procedures For the detection of BrdU or CldU or IdU, the fixed cells were prior to incubation with the antibody incubated in the following solutions: 4 N (nuclear replication) or 0.5 M (mitochondrial replication, ) HCl, 20 minutes, RT; 0.07 M NaOH, 3 minutes, RT; 20 U/ml DNase I (Sigma Aldrich), 30 minutes, 37°C .

Techniques: Mass Spectrometry, Labeling

Journal: PLoS ONE

Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

doi: 10.1371/journal.pone.0052584

Figure Lengend Snippet: A comparison of the methods based on HCl, DNase I and copper(I) ions. A ) The detection of the EU signal in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4N HCl, DNase I or with a standard two-step procedure (Cu – EU) is shown. All of the images have been acquired at 17 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – EU image is shown in the image labeled as Cu – BrdU (the acquisition time was 230 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm. B ) The detection of polyadenylated RNA in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), in cells treated with a standard two-step procedure (Cu) and in cells treated with 4 N HCl (HCl) is shown. Polyadenylated RNA was detected according to [29] . Bar: 20 µm.

Article Snippet: Alternative procedures For the detection of BrdU or CldU or IdU, the fixed cells were prior to incubation with the antibody incubated in the following solutions: 4 N (nuclear replication) or 0.5 M (mitochondrial replication, ) HCl, 20 minutes, RT; 0.07 M NaOH, 3 minutes, RT; 20 U/ml DNase I (Sigma Aldrich), 30 minutes, 37°C .

Techniques: Mass Spectrometry, Labeling

Journal: PLoS ONE

Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

doi: 10.1371/journal.pone.0052584

Figure Lengend Snippet: A comparison of the methods based on HCl, DNase I and copper(I) ions. A ) The detection of SC35 protein in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – SC35) is shown. All of the images have been acquired at 7 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – SC35 image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm. B ) The detection of coilin protein in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – coilin) is shown. All of the images have been acquired at 4 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – coilin image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm.

Article Snippet: Alternative procedures For the detection of BrdU or CldU or IdU, the fixed cells were prior to incubation with the antibody incubated in the following solutions: 4 N (nuclear replication) or 0.5 M (mitochondrial replication, ) HCl, 20 minutes, RT; 0.07 M NaOH, 3 minutes, RT; 20 U/ml DNase I (Sigma Aldrich), 30 minutes, 37°C .

Techniques: Mass Spectrometry, Labeling

Journal: PLoS ONE

Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

doi: 10.1371/journal.pone.0052584

Figure Lengend Snippet: A comparison of the methods based on HCl, DNase I and copper(I) ions. A ) The detection of H1.2 protein in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – H1.2) is shown. All of the images have been acquired at 20 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – H1.2 image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm. B ) The detection of DNA in non-treated cells (control; the cells were just fixed and permeabilized without any additional treatment), cells treated with 4 N HCl, DNase I or with a standard two-step procedure (Cu – DAPI) is shown. All of the images have been acquired at 9 ms. A simultaneous detection of the BrdU-labeled newly replicated DNA in copper-treated cells from the Cu – DAPI image is shown in the image labeled as Cu – BrdU (the acquisition time was 99 ms). The graph shows the relative signal intensities of particular kinds of the treatments used. Bars: 20 µm.

Article Snippet: Alternative procedures For the detection of BrdU or CldU or IdU, the fixed cells were prior to incubation with the antibody incubated in the following solutions: 4 N (nuclear replication) or 0.5 M (mitochondrial replication, ) HCl, 20 minutes, RT; 0.07 M NaOH, 3 minutes, RT; 20 U/ml DNase I (Sigma Aldrich), 30 minutes, 37°C .

Techniques: Mass Spectrometry, Labeling

Journal: PLoS ONE

Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

doi: 10.1371/journal.pone.0052584

Figure Lengend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

Article Snippet: Alternative procedures For the detection of BrdU or CldU or IdU, the fixed cells were prior to incubation with the antibody incubated in the following solutions: 4 N (nuclear replication) or 0.5 M (mitochondrial replication, ) HCl, 20 minutes, RT; 0.07 M NaOH, 3 minutes, RT; 20 U/ml DNase I (Sigma Aldrich), 30 minutes, 37°C .

Techniques: Labeling, Mass Spectrometry, Incubation, Activity Assay

Journal: PLoS ONE

Article Title: Binding to the Minor Groove of the Double-Strand, Tau Protein Prevents DNA from Damage by Peroxidation

doi: 10.1371/journal.pone.0002600

Figure Lengend Snippet: DNA hydrolysis with DNase I in the presence of tau protein. Tau protein and pEGFP-N1 DNA (quantitative ratio, 1/8) were incubated in 20 mM Tris-HCl buffer containing 2 mM MgCl 2 (pH 8.3, RT, 30 min), and then DNase I (0.05 units) was used to hydrolyze DNA (4730 bp, 100 ng) at 37 °C. Aliquots were taken for agarose gel electrophoresis at different time intervals as indicated. Five mM EDTA (final concentration) was employed to stop the enzymic reaction (lanes 15–20). Hydrolyses of DNA alone (lanes 1–6) and DNA in the presence of BSA (lanes 8–13) were carried out as controls.

Article Snippet: Hydrolysis of DNA with DNase I in the presence of tau protein Tau protein and pEGFP-N1 DNA (quantitative ratio, 1/8) were incubated in 20 mM Tris-HCl buffer, containing 2 mM MgCl2 , (pH 8.3, RT, 30 min), and then DNase I (0.05 units, Sigma, USA) was used to hydrolyze DNA (4730 bp, 100 ng) at 37 °C.

Techniques: Incubation, Agarose Gel Electrophoresis, Concentration Assay

Journal: Frontiers in plant science

Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

doi: 10.3389/fpls.2011.00009

Figure Lengend Snippet: TUNEL staining in necrotic spots . Transmitted light images (A,E,I,M) , sytox orange nuclear staining (B,F,J,N) and TUNEL staining (C,G,K,O) on cross-sections of 5-week-old wild-type (A–D) , and sns-D leaves (I–L) . As positive controls, DNase I-treated cross-sections of wild-type (E–H) and sns-D leaf (M–P) are shown. (D,H,L,P) are merged images of (B) with (C), (F) with (G), (J) with (K), (N) with (O) , respectively. TUNEL-positive nuclei are indicated by arrows. The bar is 25 μm.

Article Snippet: Residual DNA was removed from the RNA samples with DNase I (Ambion) in the presence of the RNase inhibitor RNasin (Promega). cDNA was produced on 1 μg of RNA using iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad).

Techniques: TUNEL Assay, Staining

Journal:

Article Title: The Expression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor by Regulatory Macrophages

doi: 10.4049/jimmunol.0802703

Figure Lengend Snippet: Changes in DNase I accessibility at the HB-EGF promoter. BMMϕs were stimulated with LPS plus IC for 0, 15, 30, 60, or 120 min and then fixed with paraformaldehyde. Nuclei were isolated and treated with DNase for 1 h on ice. DNA was then purified

Article Snippet: TRIzol reagent and DNase I were purchased from Invitrogen Life Technologies.

Techniques: Isolation, Purification

Journal: BioMed Research International

Article Title: Neutrophil Extracellular Traps Are Pathogenic in Ventilator-Induced Lung Injury and Partially Dependent on TLR4

doi: 10.1155/2017/8272504

Figure Lengend Snippet: DNase I treatment abolished NET formation in vivo. (a) The DNase I pretreated mice had a significantly decreased level of Cit-H3 expression in the lung homogenates by a Western analysis compared to the vehicle group. (b) The ratio of the Cit-H3/GAPDH intensity was significantly lower in the DNase group compared to the vehicle groups. (c) The DNase treated mice produced a lower level of BALF DNA compared to the vehicle group. ∗ P

Article Snippet: Discussion In this study, we illustrated the following: ventilation with high-tidal volume causes lung injury and inflammation in mice; NETs increase in the lung of the VILI animal model upon the elevation of known NET-associated proteins and extracellular DNA; pretreatment of NET degradation with DNase I alleviates the preceding lung injury, which means that NET formation has an adverse effect on the development of VILI; and TLR4 is involved in NET formation in VILI in mice.

Techniques: In Vivo, Mouse Assay, Expressing, Western Blot, Produced

Journal: BioMed Research International

Article Title: Neutrophil Extracellular Traps Are Pathogenic in Ventilator-Induced Lung Injury and Partially Dependent on TLR4

doi: 10.1155/2017/8272504

Figure Lengend Snippet: DNase attenuates lung injury and inflammation in mice. (a) The lung wet/dry weight ratio decreased partially in the DNase group compared with the vehicle group. (b)(c) The BALF total protein concentration and the neutrophil counts in the BALF all decreased in the DNase I pretreated mice (versus vehicle control). (d)(e) DNase I also reduced the BALF levels of IL-6 and TNF- α . (f) The lung injury score in the vehicle group was markedly higher than those in the DNase group. (g) Low (magnification 200x, scale bar = 40 μ m) and high power (magnification 400x, scale bar = 20 μ m) views of the lungs of the vehicle group and the DNase group. Hematoxylin and eosin stain. The vehicle group had much more inflammatory cell infiltration, alveolar septal thickening, pulmonary edema, and hemorrhage than the DNase group. ∗ P

Article Snippet: Discussion In this study, we illustrated the following: ventilation with high-tidal volume causes lung injury and inflammation in mice; NETs increase in the lung of the VILI animal model upon the elevation of known NET-associated proteins and extracellular DNA; pretreatment of NET degradation with DNase I alleviates the preceding lung injury, which means that NET formation has an adverse effect on the development of VILI; and TLR4 is involved in NET formation in VILI in mice.

Techniques: Mouse Assay, Protein Concentration, H&E Stain

Journal: The Journal of Cell Biology

Article Title: Chromatin Degradation in Differentiating Fiber Cells of the Eye Lens

doi:

Figure Lengend Snippet: In situ electrophoresis of an E15 lens slice. ( A ) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms ( arrowheads ) represents the OFZ. The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. ( B ) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all of the fiber cell nuclei. ( C ) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately adjacent to the OFZ. ( D ) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: ( A ) 500 μm; ( B ) 250 μm; ( C ) 100 μm; ( D ) 25 μm.

Article Snippet: To control for this, some slices were preincubated in DNase I to cause extensive DNA fragmentation in vitro.

Techniques: In Situ, Electrophoresis, Staining, Blocking Assay, Stripping Membranes

Journal: The Journal of Cell Biology

Article Title: Chromatin Degradation in Differentiating Fiber Cells of the Eye Lens

doi:

Figure Lengend Snippet: Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green and positively labeled nuclei (containing fragmented DNA) are shown in red. ( A ) At the border of the OFZ, the nuclei lose their regular shape ( arrows ) and collapse into condensed structures that are strongly labeled by the TdT assay ( arrowheads ). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. ( B ) Cortical fiber cells from a lens slice that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that the labeling is strongest immediately beneath the nuclear membrane. ( C ) Equatorial region of a lens slice that had been incubated with CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3′PO 4 termini (see text for details). ( D ) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with 3′-PO 4 termini. Bars: ( A ) 50 μm; ( B ) 10 μm; ( C ) 50 μm; ( D ) 50 μm.

Article Snippet: To control for this, some slices were preincubated in DNase I to cause extensive DNA fragmentation in vitro.

Techniques: Labeling, Incubation

Journal: Journal of Bacteriology

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC

doi: 10.1128/JB.01438-13

Figure Lengend Snippet: DNase I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).

Article Snippet: An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ).

Techniques: Footprinting, Binding Assay, Labeling, Incubation, Purification, Sequencing

Journal: Scientific Reports

Article Title: Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis

doi: 10.1038/s41598-017-01705-4

Figure Lengend Snippet: MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based DNase I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).

Article Snippet: After incubation for 30 min at 25 °C, 10 µl solution containing about 0.015 units DNase I (Promega) and 100 nM freshly prepared CaCl2 was added and further incubated for 1 min at 25 °C.

Techniques: Sequencing, Polymerase Chain Reaction, Amplification, Migration, Labeling, Footprinting, Incubation, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis

doi: 10.3389/fmicb.2018.01398

Figure Lengend Snippet: DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.

Article Snippet: The chromosomal DNA contamination of RNA samples was removed by adding DNase I (TaKaRa, Japan).

Techniques: Footprinting, Sequencing

Journal: Nature methods

Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

doi: 10.1038/NMETH888

Figure Lengend Snippet: Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three DNase I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).

Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Concentration Assay

Journal: Nature methods

Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

doi: 10.1038/NMETH888

Figure Lengend Snippet: Location of DNase I hypersensitive sites relative to the annotated genome ( a,b ) DNase-chip peaks were mapped to ENCODE regions stratified by gene density and human-mouse sequence conservation for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). ( c ) The genomic locations of DNase I hypersensitive sites (detected with at least two concentrations of DNase I) and computationally generated random controls ( n = 1,000) were compared to Gencode transcription start and end sites (within a 2-kb window), CpG islands, first introns, non-first introns, first exons, non-first exons, conserved sequences (MCS), conserved sequences minus coding exons (MCS-no-CDS). The number of DNase I hypersensitive sites at different distances (0 kb, 2 kb, 10 kb, and 25 kb) from Gencode genes was also determined. Error bars represent the entire range of values seen randomly generated mock datasets ( n = 1,000). Compared to the random controls, the locations of the DNase-chip peaks are significantly (Monte Carlo P

Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

Techniques: Chromatin Immunoprecipitation, Sequencing, Generated

Journal: Nature methods

Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

doi: 10.1038/NMETH888

Figure Lengend Snippet: Identification of cell type–specific DNase I hypersensitive sites ( a,b ) Real-time PCR was performed on both CD4 + T cells and GM06990 cells. Real-time PCR using primer sets that flank random regions of the genome or DNase-chip peaks that are present for both cell types ( a ). Real-time PCR using primer sets that flank DNase-chip peaks that are present in only CD4 + T cells (CD4) or GM06990 cells (GM; b ).

Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Nature methods

Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

doi: 10.1038/NMETH888

Figure Lengend Snippet: Expression of genes relative to proximity to DNase I hypersensitive sites ( a,b ) The distance of each transcription start site (blue dots) to the nearest DNase I hypersensitive site was compared to the gene expression values of each transcript for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). Horizontal red lines mark the expression level that separates most genes that have a DNase I hypersensitive site nearby (

Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

Techniques: Expressing

Journal: Nature methods

Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

doi: 10.1038/NMETH888

Figure Lengend Snippet: DNase-chip protocol ( a ) Pulsed field gel electrophoresis of DNase I–digested nuclear DNA. The concentrations of DNase I used for DNase-chip are labeled as A, B and C. ( b ) Outline of DNase-chip protocol. ( c ) Histogram of signal ratios of DNase I–treated versus random-sheared DNA. Tiled oligos that displayed the top 5% ratios are located to the right of the red bar. ( d ) Identification of regions with significant P values. The raw ratio data are plotted in gray, with the y-axis label on the right; the top 5% cutoff is displayed as a dotted horizontal gray line. The P value data for sliding 500-bp windows are plotted in red, with the y -axis label on the left.

Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

Techniques: Chromatin Immunoprecipitation, Pulsed-Field Gel, Electrophoresis, Labeling

Journal: Nucleic Acids Research

Article Title: Heat shock factor 1 promotes TERRA transcription and telomere protection upon heat stress

doi: 10.1093/nar/gkx208

Figure Lengend Snippet: TERRA accumulation requires HSF1 upon heat shock. ( A ) Western blot analysis of HSF1 WT and KD unstressed and stressed cells. Tubulin is shown as a loading control. ( B ) Dot blot analysis of TERRA. Total RNA was extracted from WT and HSF1 KD cells before and after HS and treated with DNAse. Various amount of total RNA fractions were subjected to RNA dot blot analysis using a radiolabelled TERRA probe. RNA were treated with RNase in parallel.The same blot was also hybridized after staining with a control probe specific for U2 transcripts. ( C ) Quantification of TERRA and U2 levels in WT and HSF1 KD cells before and after HS normalized with 37°C conditions. Standard deviation (SD) was calculated from 3 independent experiments. P -value = 0.0213 ( D ) Representative confocal images of FISH analyses of TERRA expression in WT and HSF1 KD cells before and after HS. RNase treatment induces an elimination of TERRA signal. Scale bar: 10 μm. ( E ) 3D quantification analysis of mean TERRA foci volume in the different conditions tested by RNA FISH using volocity software. The P -values calculated by unpaired Student's t -test with Welch's corrections at 37°C and at 43°C for WT and HSF1 KD cells are respectively P

Article Snippet: RNA was treated with DNase (Ambion) for 30 min at 37°C.

Techniques: Western Blot, Dot Blot, Staining, Standard Deviation, Fluorescence In Situ Hybridization, Expressing, Software

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

doi: 10.1038/s41598-017-16932-y

Figure Lengend Snippet: Majority of indels is present between both cleavage sites after editing with Cas9 nickase. Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.

Article Snippet: Cas9 nuclease The two sgRNAs used with the Cas9 nickase were individually cloned into the pX330 plasmid (Addgene Plasmid #42230).

Techniques: Sequencing

Journal: DNA and Cell Biology

Article Title: Regulation of Apoptotic Endonucleases by EndoG

doi: 10.1089/dna.2014.2772

Figure Lengend Snippet: Both EndoG and bleomycin induce DNase I and DNase X transcription in isolated rat brain cell nuclei. (A, B) In vitro transcription of DNase I and DNase X genes in isolated rat brain nuclei exposed with varying concentrations of recombinant EndoG (recEndoG)

Article Snippet: Primary antibodies, anti-DNase I, anti-CAD, and anti-GAPDH were detected with anti-rabbit IgG-horseradish peroxidase (HRP) while anti-DNase X, anti-DNase I-like 2, and anti-DNase γ were detected with donkey anti-goat IgG-HRP using SuperSignal chemiluminescent kit (Pierce Biotechnology).

Techniques: Isolation, In Vitro, Recombinant

Journal: DNA and Cell Biology

Article Title: Regulation of Apoptotic Endonucleases by EndoG

doi: 10.1089/dna.2014.2772

Figure Lengend Snippet: Induction of DNase I-like endonucleases in EndoG-overexpressing cells. (A) Western blotting for DNase I, DNase X (both at 8 h), DNase 1-like 2, DNase γ (both at 12 h), caspase-activated DNase (CAD) (at 8 h post-transfection),

Article Snippet: Primary antibodies, anti-DNase I, anti-CAD, and anti-GAPDH were detected with anti-rabbit IgG-horseradish peroxidase (HRP) while anti-DNase X, anti-DNase I-like 2, and anti-DNase γ were detected with donkey anti-goat IgG-HRP using SuperSignal chemiluminescent kit (Pierce Biotechnology).

Techniques: Western Blot, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis

doi: 10.1073/pnas.1708247114

Figure Lengend Snippet: Endothelial damage and complement activation by ANCA-induced NET generation. ( A ) HUVECs were coincubated with NETs isolated from either TNFα-primed human neutrophils without further stimulation (unstim), isotype mAb-stimulated (ctrl), or αMPO mAb-stimulated (αMPO) neutrophils. Permeability of the EC monolayer was measured after 1 h using albumin-FITC. NET degradation by DNase protected from endothelial damage. ( B ) Human neutrophil preincubation with Nec-1 protected from NET-induced endothelial damage. ( C ) NETs were coincubated in the absence (w/o) or presence of human serum and stained after PFA fixation with PI (DNA; red) and αC3d antibody (FITC; green). Unstimulated cells and isotype-stimulated neutrophils displayed no NETs and no C3d deposition, whereas αMPO mAb-stimulated cells coincubated with serum showed NET formation with strong C3d costaining. DNase pretreatment prevented C3d staining. A typical example is depicted. ( D ) NETs were coincubated with different human sera as indicated and stained after fixation with PI (DNA; red) and αC3d antibody (FITC; green). Isotype-stimulated neutrophils displayed no NETs and no C3d deposition; αMPO mAb-stimulated neutrophils displayed NET formation and—when coincubated with serum—C3d deposition. C3d deposition was not affected by C1q deficiency but strongly reduced in both C3-deficient (C3-def) and complement factor B-deficient (FB-def) sera. ( E ) αMPO mAb-induced NETs that were coincubated with human serum caused complement activation with C5a generation. The effect was prevented by NET degradation using DNase I. ( F ) Increased EC permeability by αMPO mAb-induced NETs is mediated by the alternative complement pathway, as both C3-deficient and complement factor B-deficient sera provided protection whereas C1q deficiency did not. Error bars indicate means ± SEM. Comparisons were made using t test or ANOVA; * P

Article Snippet: C1q-deficient and factor B-deficient sera were from Quidel, C3-deficient serum was from Sigma, propidium iodide, BSA-FITC, PMA, necrostatin-1, and recombinant DNase I were from Sigma-Aldrich, necrostatin-1s was from VWR, necrosulfonamide was from Millipore, and Sytox green was from Invitrogen.

Techniques: Activation Assay, Isolation, Permeability, Staining

Journal: Frontiers in Immunology

Article Title: d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

doi: 10.3389/fimmu.2017.00975

Figure Lengend Snippet: d (−) lactic acid induced the release of cell-free DNA from neutrophils that activated bovine umbilical vein endothelial cell (BUVEC) sheets by ICAM display. (A) Supernatants collected from neutrophils treated with 5 mM d (−) lactic acid plus or minus DNase I were perfused onto BUVEC for 10 min. Next, fresh, untreated neutrophils were perfused on endothelial monolayers, n = 5, ** P

Article Snippet: Next, cells were stimulated with 5 mM of d (−) lactic acid with 90 U DNase I (PanReac AppliChem, Darmstadt, Germany) or with vehicle (0.01% ethanol) for 30 min at 37°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

doi: 10.3389/fimmu.2017.00975

Figure Lengend Snippet: PAD4 activation regulates d (−) lactic acid-induced neutrophil extracellular trap (NET) formation and subsequent adhesion. (A) Immunofluorescence of bovine neutrophils pre-treated with monocarboxylate transporter 1 inhibitor (Ar-c155858), PAD4 inhibitor (Cl-amidine), or vehicle (0.01% DMSO) for 60 min, followed by stimulation with either 5 mM d (−) lactic acid or vehicle concomitant with DNase I (90 U) or controls for 30 min. Neutrophils were probed with anti-H 4 citrullinated 3/alexa 405, CD11b/alexa 635, and PicoGreen as a probe for DNA; representative images from three independent experiments, scale bar: 50 µm. (B) Fold of control [(number of NETs/number of neutrophils) × 100] from neutrophils treated with 1 µM Ar-c155858, 200 µM Cl-amidine or vehicle for 1 h and stimulated with 5 mM of d (−) lactic acid or vehicle (control) together or not with DNAse I (90 U) for 30 min, n = 3. *** P

Article Snippet: Next, cells were stimulated with 5 mM of d (−) lactic acid with 90 U DNase I (PanReac AppliChem, Darmstadt, Germany) or with vehicle (0.01% ethanol) for 30 min at 37°C.

Techniques: Activation Assay, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

doi: 10.3389/fimmu.2018.00968

Figure Lengend Snippet: Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

Techniques: Inhibition, Hi-C, Derivative Assay, Fluorescence, Negative Control, Positive Control

Journal: Frontiers in Immunology

Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

doi: 10.3389/fimmu.2018.00968

Figure Lengend Snippet: Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

Techniques: Incubation, Derivative Assay