Journal: The EMBO Journal

Article Title: Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration

doi: 10.1038/emboj.2012.219

Figure Lengend Snippet: Endo III blocks XerC-catalysis on pseudo-HJ and displaces it from them. ( A ) Scheme of the suicide pseudo-HJ indicating the mismatch engineered to slow down re-ligation after XerC-cleavage. KMnO 4 sensitive residues in the XerC- and XerD-binding att P(+) arms are indicated by a star. ( B ) Resolution of att P(+)/ dif suicide pseudo-HJs. Legend as in Figure 4D . ( C ) DNase I protection and ( D ) KMnO 4 sensitivity assays of the att P(+)/ dif 1 pseudo-HJ substrate. The analysed strand was labelled on its 5′ end. A scheme of the analysed strand is drawn on the left of the gels. KMnO 4 sensitive residues in the XerC- and XerD-binding sites are indicated by a star.

Article Snippet: E. coli Endo III, T7 endo I and Dnase I were purchased from New England Biolabs.

Techniques: Ligation, Binding Assay

Journal: The Journal of General Virology

Article Title: Marek's disease virus infection of phagocytes: a de novo in vitro infection model

doi: 10.1099/jgv.0.000763

Figure Lengend Snippet: Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control (DNase-treated RNA); D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).

Article Snippet: RT-PCR RNA samples were extracted using RNeasy Mini Kits (Qiagen) and treated with DNase (Ambion Turbo DNA-free Kits, Life Technologies).

Techniques: Infection, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

Journal: RNA

Article Title: Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

doi: 10.1261/rna.2314406

Figure Lengend Snippet: DRBM2 is essential for RNA editing by ADAR2. N2A cells were cotransfected with expression plasmids encoding an editing construct, pQR1, encompassing the Q/R site of the rat GluR2 pre-mRNA in a minimal hairpin and the EGFP-ADAR2 fusion constructs schematically represented. A dot indicates a mutation; a line indicates a deletion ( A ). Forty-eight after transfection, RNA was purified, DNase treated, and subjected to RT-PCR. ( B ) The editing level at the Q/R-site was determined by primer extension. The lower band represents unextended primer, P; the middle band represents cDNA from unedited transcripts, U; and the top band represents cDNA from transcripts edited at the Q/R site, E. In the lane marked pQR1 , the plasmid encoding the editing substrate was used for the primer extension. ( C ) The U and E signals were quantitated by PhosphorImaging, and the level of editing was determined as the intensity of E relative to the sum of the E and U intensities. The average and standard deviation of the results from a representative experiment performed in triplicate are shown. ( D ) Fluorescence microscope images (Zeiss Axiovert200) of living N2A cells were taken 24 h after transfections with the indicated EGFP-ADAR2 constructs.

Article Snippet: Two days after transfection, RNA was purified with the RNeasy kit (Qiagen) and treated with DNase (Roche), and after purification, Superscript II RT (Invitrogen) was used for reverse transcription (RT) of the RNA with an SP6 primer at 42°C for 40 min followed by 10 min at 45°C.

Techniques: Expressing, Construct, Mutagenesis, Transfection, Purification, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Standard Deviation, Fluorescence, Microscopy

Journal: PLoS Pathogens

Article Title: The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses

doi: 10.1371/journal.ppat.1006031

Figure Lengend Snippet: The VP35 IID reduces Th1 responses. A. Schematic representation of the genomes of wt EBOV and mutant EBOVs used to infect cells; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted GFP gene is indicated by green rectangles. B. Schematic representation of the cultivation experiment: PBMCs from CMV-positive donors were labeled with CFSE, inoculated with the indicated viruses at MOI of 2 PFU/cell or SEB, and stimulated with CMV pp65 peptides for 4 hours. PBMCs were washed and cultured for 7 days. Thereafter, cells were re-stimulated for 6 hours in culture medium containing CMV pp65 peptides in presence of brefeldin A, monensin, anti-CD28 mab, anti-CD49d mab and DNase. As a positive control, cells were stimulated with SEB and treated with phorbol-12-myristate-13 acetate (PMA) and ionomycin. Cells were stained intracellularly for the indicated cytokines followed by multicolor flow cytometry. C. Percentages of total and proliferating (CFSE - ) CD4 + T cells secreting total IFNγ, IL-2 or TNFα normalized to wt EBOV (100%, indicated by the red horizontal lines). Percentages of proliferating and total CD4 + T cells were individually normalized to wt EBOV values in the same donor (100%, indicated by the red horizontal lines). Cells from each individual donor are represented by the same symbol across the panels; mean values are indicated by horizontal bars. Red asterisks indicate significant differences to wt EBOV (p

Article Snippet: Stimulation and staining After 7 days of culture, cells were harvested, washed and 2 x 106 cells were stimulated for 6 hours in culture medium with 10 μg/ml Brefeldin A (Sigma-Aldrich), 0.7 μg/ml monensin (GolgiStop, BD Biosciences), 1 μg/ml anti-CD28 (BD Biosciences), 1 μg/ml anti-CD49d (BD Biosciences), 20 μg/ml DNase (Calbiochem) and 2 μg/ml of 15-mer CMV pp65 peptides.

Techniques: Mutagenesis, Labeling, Cell Culture, Positive Control, Staining, Flow Cytometry, Cytometry

Journal: Infection and Immunity

Article Title: The Staphylococcus aureus KdpDE Two-Component System Couples Extracellular K+ Sensing and Agr Signaling to Infection Programming ▿ Sensing and Agr Signaling to Infection Programming ▿ † Sensing and Agr Signaling to Infection Programming ▿ † ‡

doi: 10.1128/IAI.01180-10

Figure Lengend Snippet: Identification of the KdpE-binding sequences by DNase I footprinting. (A) Identification of the KdpE-binding site on the promoter of kdpFABC by DNase I footprinting assays. The black frame indicates the DNA region protected from DNase I by KdpE. (B) KdpE-binding

Article Snippet: After incubation at 37°C for 5 min, S. aureus cells were prepared for total RNA extraction using the Trizol method (Invitrogen), and any residual DNA was removed with DNase (RNase free; TaKaRa).

Techniques: Binding Assay, Footprinting

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

Article Title: Increased cell proliferation, but not reduced cell death, induces lymphocytosis in bovine leukemia virus-infected sheep

doi: 10.1073/pnas.142100999

Figure Lengend Snippet: BrdUrd incorporation into B lymphocytes in vivo . ( A ) Three BLV-infected sheep (nos. 8, 104, and 293) and three controls (nos. 117, 1092, and 1097) were injected intravenously with 500 mg BrdUrd, and an aliquot of blood (1 ml) was collected 3 days later. After lysis of the red blood cells, B cells were labeled with biotinylated 1H4 monoclonal antibody and streptavidin-PE conjugate. Then, the cells were stained with anti-BrdUrd FITC antibody in the presence of DNase and analyzed by two-color flow cytometry ( x axis, BrdUrd; y axis, B lymphocytes). Ten thousand cells (lymphocytes, monocytes, and granulocytes) were acquired, and PBMCs were selected by the FSC/SSC gating. The total numbers of B cells are indicated in the upper quadrants. ( B ) Kinetic analysis of BrdUrd + B cells. Blood samples from six sheep (see A ) were collected at different days after BrdUrd injection. The ratio (in %) of BrdUrd + cells within the total B lymphocyte population was determined, and the data corresponding to the measured incorporation rates were fitted to a mathematical model, yielding a theoretical fit (see Materials and Methods ). Figure shows the average data for the three infected and the three control sheep. ( C ) Minimal proliferation and death rates (± SD) estimated from fitting the source model to the data deduced from two independent experiments. ( D ) Summary of the proliferation and death rates (± SD) and estimation of the population growth.

Article Snippet: Finally, leukocytes were stained with anti-BrdUrd FITC antibody in the presence of DNase (Becton Dickinson) for 30 min at room temperature and analyzed by flow cytometry.

Techniques: In Vivo, Infection, Injection, Lysis, Labeling, Staining, Flow Cytometry, Cytometry

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

Article Title: Increased cell proliferation, but not reduced cell death, induces lymphocytosis in bovine leukemia virus-infected sheep

doi: 10.1073/pnas.142100999

Figure Lengend Snippet: Proliferation and viral expression. ( A ) Two days after BrdUrd injection, PBMCs from noninfected (no. 1097), asymptomatic (no. 8), and lymphocytic (nos. 2668 and 2658) sheep were isolated and cultivated during 18 h. The cells then were fixed and incubated with anti-p24 antibody 4′G9, which recognizes the viral capsid protein, and with a PE-conjugated secondary antibody. Finally, cells were stained with anti-BrdUrd FITC with DNase and analyzed by flow cytometry. A representative experiment (out of three) is represented by dot plots (10,000 selected events). Numbers represent the percentages of positively stained cells in each quadrant. ( B ) Schematic representation of BrdUrd incorporation in vivo (hypothetical) and p24 labeling after short-term culture ( ex vivo ). Hatched areas represent the uninfected cell; black squares and full circles indicate BrdUrd and p24 markers, respectively. Crosses indicate that cells were undetectable by FACS.

Article Snippet: Finally, leukocytes were stained with anti-BrdUrd FITC antibody in the presence of DNase (Becton Dickinson) for 30 min at room temperature and analyzed by flow cytometry.

Techniques: Expressing, Injection, Isolation, Incubation, Staining, Flow Cytometry, Cytometry, In Vivo, Labeling, Ex Vivo, FACS

Journal: Frontiers in Microbiology

Article Title: A Novel Postbiotic From Lactobacillus rhamnosus GG With a Beneficial Effect on Intestinal Barrier Function

doi: 10.3389/fmicb.2019.00477

Figure Lengend Snippet: Characterization of soluble protein detected from LCS. LCS was pretreated with proteinase K, DNAse or carbohydrase, then the digested products were applied to treat Caco-2 monolayers, the soluble mucin (A) and mucin-covered Caco-2 surface ( C , upper panel) were detected using PAS assay, expression of MUC2 ( B , middle and lower panels) was evaluated by immunoblot, β-actin was used as loading control. (D) LCS was precipitated and separated by SDS-PAGE (Lane 2). Data are given as means ± SEM; ∗∗ P

Article Snippet: Then 20 μL proteinase inhibitor was added. (2) DNAase treatment: 1 ml LCS was digested with 10 μl DNAase (Tiangen Biotech Co., Ltd., Beijing, China) for 25 min at 37°C.

Techniques: PAS Assay, Expressing, SDS Page

Journal: Journal of Virology

Article Title: Replication of Oral BK Virus in Human Salivary Gland Cells

doi: 10.1128/JVI.02777-13

Figure Lengend Snippet: Significantly higher viral loads were detected for HIVSGD-1 than for HIVSGD-2 BKPyV post-HSG cell transfection. (A) The chart shows viral loads (VLs) measured from the supernatant of HSG cells transfected with BKPyV genomes. HSG cell supernatant was harvested 6 days p.t., filtered, and DNase treated, and BKPyV was quantified via qPCR and is depicted as BKPyV copies/μl. VL levels are ranked from high to low as MM, HIVSGD-1, U1, and HIVSGD-2 BKPyV. The P values were determined via one-way analysis of variance (ANOVA), and error bars represent standard deviations. (B) Representative images visualizing HIVSGD-1 and MM BKPyV virions from HSG cell supernatant via transmission electron microscopy (TEM) 6 days p.t. (black arrows).

Article Snippet: Transfected and infected HSG and Vero cell supernatants were passed through 0.45-μm-pore filters and DNase treated (Promega), removing debris/cells and nonencapsulated viral DNA, respectively.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

Journal: Journal of Virology

Article Title: Replication of Oral BK Virus in Human Salivary Gland Cells

doi: 10.1128/JVI.02777-13

Figure Lengend Snippet: HIVSGD-1 and MM BKPyV exhibited similar replication kinetics in HSG and Vero cells with increasing viral loads postinfection. (A) The chart at the top shows viral loads (VLs) in BKPyV copies/μl, measured from the supernatant of infected HSG and Vero cells, 2, 4, 6, and 8 days p.i. Filtered supernatant from transfected HSG cells was used to infect HSG (black) and Vero (white) cells at equal viral loads. Supernatant from infected cells was harvested, filtered, and DNase treated, and BKPyV VL was quantified via qPCR. BKPyV HIVSGD-1 VLs increased over 6 orders of magnitude and MM VLs over 5 orders of magnitude during the 8 days p.i. Encapsulated HIVSGD-2 DNA and U1 BKPyV DNA were not detected. Error bars represent standard deviations. Graphed VLs from infected HSG cells and Vero cells show similar growth kinetics for BKPyV HIVSGD-1 and MM. (B) HIVSGD-1 (Vero- and HSG cell-derived) and MM (Vero cell-derived) BK virions were detected under TEM 8 days p.i. MM BKPyV-infected HSG supernatant was not analyzed. No virions were detected for HIVSGD-2, U1, and mock infection in HSG or Vero cells (data not shown). Mock infections were accomplished by using supernatant from mock-transfected cells.

Article Snippet: Transfected and infected HSG and Vero cell supernatants were passed through 0.45-μm-pore filters and DNase treated (Promega), removing debris/cells and nonencapsulated viral DNA, respectively.

Techniques: Infection, Transfection, Real-time Polymerase Chain Reaction, Derivative Assay, Transmission Electron Microscopy

Journal: Scientific Reports

Article Title: Role of DNA-LL37 complexes in the activation of plasmacytoid dendritic cells and monocytes in subjects with type 1 diabetes

doi: 10.1038/s41598-020-65851-y

Figure Lengend Snippet: The confirmation of DNA-LL37 complexes formation by EMSA and confocal microscopy. LL37 was incubated with self-DNA at different ratios and time points. ( a ) Electrophoretic mobility shift assay (EMSA) of DNA-LL37 complexes was performed. Lanes 2 and 3 show the formation of complexes. ( b ) DNA alone (control, Lane 1), DNA complex treated with DNase 1 (lane 2) and untreated complex (lane 3). ( c ) EMSA on native PAGE gel shows the stable formation of self-DNA complexes with LL37 (lane 2), whereas self-DNA did not form complexes with other beta cell associated proteins, such as GAD65 (lane 4) and IA-2 (lane 6), ( d ) PAGE gel shows the formation of DNA-LL37 complexes (lane 1) and LL37 alone (Lane 2), ( e ) Confocal image of Hoechst labelled DNA alone and ( f ) Hoechst labelled DNA-LL37 complex, forming visible aggregates, confirming complex formation. ( g ) DNA-LL37 complexes were treated with DNase I to remove extra-unbound DNA. The exposure time was same in all procedures and images. The gel images are of single gel and do not contain cropped or cut-outs from other gels. Gels ( a, b and d ) are cropped from their respective gels to increase the clarity and conciseness to include only relevant lanes (full gel images are provided in supplementary figures S1 , S2 and S3 ).

Article Snippet: Free DNA was removed by DNase 1 (4 units in 2 µL, Sigma-Aldrich, USA) treatment for 30 minutes at RT and the reaction was stopped with 1 M NaCl.

Techniques: Confocal Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Clear Native PAGE, Polyacrylamide Gel Electrophoresis

Journal: PLoS ONE

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

doi: 10.1371/journal.pone.0028537

Figure Lengend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Article Snippet: RNA was extracted from L929 cells using Trizol (Invitrogen) according to the manufacturer's instructions, followed by DNAse treatment using DNA-free (Ambion) according to manufacturer's instructions.

Techniques: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Immunity

Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

doi: 10.1016/j.immuni.2018.01.013

Figure Lengend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker