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

Journal: Science Advances

Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

doi: 10.1126/sciadv.1600502

Figure Lengend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

Article Snippet: RNase-free DNase was obtained from Qiagen.

Techniques: Fluorescence

Journal: Cells

Article Title: Liver Immune Cells Release Type 1 Interferon Due to DNA Sensing and Amplify Liver Injury from Acetaminophen Overdose

doi: 10.3390/cells7080088

Figure Lengend Snippet: DNase treatment after hepatic injury induction. ( A ) Scheme showing APAP treatment time points (600 mg/kg) and DNase (1000 U/L) in mice over time. ( B ) ALT serum levels to assess hepatic injury at 12 and 24 h after DNase treatment. ( C – G ) Gene expression of different sensors in hepatic non-parenchymal cells between treated and non-treated groups with DNase. Cells were collected 12 h after administration of APAP (600 mg/kg) and relative expression was done using control NPCs (saline) as a reference. ( H – K ) Gene expression of different sensors in hepatocytes between DNase-treated and non-treated groups. ( K ) Flow cytometry to evaluate IFN-I production by non-parenchymal liver cells in DNase-treated and untreated groups. Cells were collected from IFN YFP/YFP mice 12 h after administration of APAP (600 mg/kg). (Mean ± SEM; n = 4); * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. The relative gene expression was done using saline hepatocytes as a reference.

Article Snippet: To confirm that extracellular DNA released during necrosis was fuelling liver NPC activation and hepatocyte necrosis, we treated mice during the evolution of APAP-induced injury with a commercially available DNase (Sigma-Aldrich) ( A).

Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry

Journal: PLoS ONE

Article Title: Measles Virus Matrix Protein Inhibits Host Cell Transcription

doi: 10.1371/journal.pone.0161360

Figure Lengend Snippet: MeVM localizes to the nucleus by associating with chromatin in transfected cells. A. Sub-confluent overnight cultures of COS-7 cells grown on glass coverslips were transfected with pEPI-DESTC-GFP-M as in Fig 1 and fixed at 24 h p.t., (row 1, labeled “no add”), permeabilised with NP-40 (row 2, labelled “NP40”), and treated with DNAse 1 (row 3, labelled “NP40+DNAse 1”). Cells were probed with mouse anti-Lamin B1 antibody, followed by Alexa-568 conjugated secondary antibody and coverslips mounted on glass slides using ProLong Gold Antifade with DAPI. Green (GFP-MeVM), blue (DAPI) and red (Lamin) fluorescence was analysed by CLSM; images were processed with ImageJ to generate the merged images shown in the far right column. Scale bar = 5 μm. B. Sub-confluent overnight cultures of COS-7 cells grown on glass coverslips were transfected with pEPI-DESTC-GFP and fixed at 24 h p.t., (row 1, labeled “no add”) and permeabilised with NP-40 (row 2, labelled “NP40”), Coverslips were mounted on glass slides using ProLong Gold Antifade with DAPI. Green (GFP), and blue (DAPI) fluorescence was analysed by CLSM; images were processed with ImageJ to generate the merged images shown in the far right column. Scale bar = 5 μm.

Article Snippet: In separate experiments the transcription products were treated with DNase 1 (Sigma, 0.1 mg/ml) or RNase 1 (Sigma, 0.1 mg/ml) or proteinase k (Invitrogen, 0.2 mg/ml) for 1 h at 37°C prior to gel electrophoresis as above.

Techniques: Transfection, Labeling, Fluorescence, Confocal Laser Scanning Microscopy

Journal: European Journal of Microbiology & Immunology

Article Title: Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin

doi: 10.1556/1886.2018.00026

Figure Lengend Snippet: Semi-quantitative assay in gel electrophoresis for the evaluation of DNase activity. 1 μg of DNA (amplicon atr ) incubated with 10 μl of S. agalactiae culture supernatant (NEM316 vs CC19 clinical strain) for 1h, 2h, 4h, overnight at 37°C. Neg, Negative control: without culture supernatant; ON, Overnight; M, Molecular weight Ladder.

Article Snippet: Qualitative evaluation of DNase production was performed for all S. agalactiae strains; this was done by inoculation of DNA–methyl green agar plates (Oxoid, Basingstoke, England) or DNase test agar with toluidine blue plates (Sigma-Aldrich, USA).

Techniques: Nucleic Acid Electrophoresis, Activity Assay, Amplification, Incubation, Negative Control, Molecular Weight

Journal: Pain

Article Title: The bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease

doi: 10.1097/j.pain.0000000000001225

Figure Lengend Snippet: MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The RNA was isolated, DNase treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p

Article Snippet: RNA was isolated from whole spinal cords using Trizol and was DNAse treated (Invitrogen, Amsterdam) and was converted into cDNA using oligo(dT)12–18 primers with the Advantage RT for PCR Kit (Takara).

Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Experimental Cell Research

Article Title: Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation

doi: 10.1016/j.yexcr.2018.05.031

Figure Lengend Snippet: Comparison of uroplakin and urothelium differentiation-associated transcription factor gene expression by NHB and NHU cell cultures. Employing protocols developed to differentiate NHU cells by PPARγ activation, cell cultures of NHB or NHU cells were exposed to 1 µM troglitazone and 1 µM PD153035 (TZ/PD) for 24 h, maintained in 1 µM PD153035 and harvested at 12, 24, 48 and/or 72 h. Control cultures were exposed to vehicle (0.1% DMSO) alone. (A) RTqPCR for three independent NHB cell lines (represented by different symbols), versus a single NHU cell line for comparison of uroplakin (UPK1A, UPK1B, UPK2, UPK3A and UPK3B) mRNA expression at the 72 h time-point. All data has been normalised to GAPDH expression and is presented relative to the DMSO-treated NHB cells for each gene except UPK3A, where the data is shown relative to the DMSO treated NHU cells due to absent UPK3A gene expression by NHB cells. BLOD = Below Limit of Detection. Statistical analysis was performed using a two-tailed, paired t -test to determine whether TZ/PD resulted in any significant change in gene expression in NHB cells. *represents P ≤ 0.05, ** represents P ≤ 0.01. Error bars represent standard deviation. (B-C) RT-PCR of ELF3, FOXA1, GATA3, GRHL3, IRF1, KLF5 and PPARG mRNA expression by (B) NHB cells and (C) NHU cells. RNA was extracted at the 12, 24 and 48 h time-points and then DNAase-treated and used to generate cDNA for RT-PCR. GAPDH was used as an internal loading control. A no-template (H 2 O) control was included as a negative control for the PCR reaction, and genomic DNA was used as the positive control (+ctrl). No product was amplified from RT-negative controls (not shown). Experiments were performed on n = 2 independent NHB donor cell lines and representative results shown.

Article Snippet: Any contaminating genomic DNA was removed by DNase digestion (DNA-free™, Ambion) and checked using RT-negative controls. cDNA was synthesized from 1 µg of total RNA using the first-strand synthesis system primed with random hexamers (Invitrogen).

Techniques: Expressing, Activation Assay, Two Tailed Test, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Positive Control, Amplification

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: Journal of Virology

Article Title: Cellular Cholesterol Facilitates the Postentry Replication Cycle of Herpes Simplex Virus 1

doi: 10.1128/JVI.00445-17

Figure Lengend Snippet: Cholesterol depletion affects the synthesis of viral proteins and the yield and release of infectious HSV-1. (A and B) Vero cells were infected with HSV-1 KOS (MOI = 5) for 1 h at 37°C. The cultures were mock treated (−) or treated with 50 mM MβCD (+) for 45 min at room temperature and then rinsed and replenished with serum-free medium. At 19 h p.i, (A) or the indicated times (B), lysates were separated by SDS-PAGE. The Western blots were probed with anti-HSV polyclonal antibody (A) or monoclonal antibodies to tubulin, ICP4, ICP8, and VP16 (B). (C to E) Densitometry of the HSV-1 proteins in panel B presented as the ratio of viral protein to tubulin. (F) Supernatant fractions or complete whole-cell lysates were collected at 19 h p.i., and viral titers were determined. The data are means of triplicate determinations with standard deviations and are representative of the results of at least three independent experiments. Numerical percentages of extracellular infectious HSV-1 are indicated. (G) DNA was extracted from infected cells that were treated with DNase or left untreated. Cell-associated HSV-1 genome copies were quantitated by qPCR. The data presented are means of the results of three independent experiments. Numerical percentages of copy numbers are indicated to the right of the bars. Student's t test; DNase resistance in untreated versus MβCD-treated cells; P = 0.032.

Article Snippet: Samples were treated with 2 μg/ml DNase (Bio-Rad), and viral genomic DNA was extracted using a QIAamp Blood DNA kit (Qiagen, Valencia, CA).

Techniques: Infection, SDS Page, Western Blot, Real-time Polymerase Chain Reaction

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 ONE

Article Title: Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

doi: 10.1371/journal.pone.0029806

Figure Lengend Snippet: Synergistic effects of TLR ligands with poke weed lectin. A: Effect of TLR2 blockage on human B cell proliferation in response to PWM. CD19+ human peripheral blood B cells were labelled with CFSE, preincubated with anti-TLR2 mAb or the corresponding isotype control (murine IgG 1κ ), and stimulated with Pam 3 CSK 4 or PWM (10 µg/ml) ± SpA (5 µg/ml) for 5 days. Proliferation was assessed by CFSE dilution. The gate denotes the percentage of live gated proliferating B cells as depicted. One representative experiment of n = 3 is shown. B: Synergistic effects of PWM and immunostimulatory DNA. Human B cells were stained with CFSE for assessment of B cell proliferation. Stimulation was performed with CpG ODN 2006 (PTO), DNAse- or mock (reaction buffer only)-treated PWM, 2006 GC (PTO) or CpG 2006 (PO) and combinations thereof as indicated. The graphs show the results from one representative experiment from n≥3. The percentage of proliferating B cells is indicated in the graphs.

Article Snippet: For DNAse treatment 20 µl PWM stock 0.1 mg/ml were incubated with or without 1 unit RNAse-free DNAse (Roche, Mannheim, Germany) and 2 µl 10× incubation buffer provided by the manufacturer over night at 37°C.

Techniques: Staining

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: An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

doi:

Figure Lengend Snippet: Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 μl of each 25-μl reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.

Article Snippet: Total RNA was then treated with 1 μl (10 to 50 U) of DNase (RNase free; Boehringer Mannheim) per 10 μg of RNA for 30 min at 37°C in the presence of 5 mM MgCl2 , with subsequent inactivation at 65°C for 10 min in the presence of 5 mM EDTA; RNA concentration was calculated based on the optical density at 260 nm.

Techniques: Expressing, Agarose Gel Electrophoresis, Plasmid Preparation, Negative Control

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: Fertility and sterility

Article Title: Is anti-mullerian hormone a marker of acute cyclophosphamide-induced ovarian follicular destruction in mice pretreated with cetrorelix?

doi: 10.1016/j.fertnstert.2011.04.008

Figure Lengend Snippet: Cetrorelix reduced apoptosis in the granulosa cells of mice treated with cyclophosphamide. (A–F) Immunofluorescent detection of apoptotic cells (stained yellow) by TUNEL in ovaries exposed to saline (Con), antagonist alone (Ant), cyclophosphamide (Cyc) or pretreated with antagonist (Cyc + Ant). (G) The positive control (Positive Con) was incubated with DNASE 1, and (H) the negative control (Negative Con) was incubated with fluorescein-labeled dUTP. Scale bar = 100µm, magnification= 20×. (I) Quantitative comparison of mean TUNEL scores of the ovaries receiving cyclophosphamide (100mg/kg) with and without antagonist. Percentage of apoptotic cells (mean ± SEM) in the cyclophosphamide-only (Cyc100) treated group vs the cyclophosphamide and antagonist group (Cyc100 + Ant), respectively (19% ± 6 vs 6% ± 6, p=0.0033, t-test). Each bar represents 3 mice.

Article Snippet: A positive control was created by incubating one section in DNASE 1 (Ambion) for 10 minutes at room temperature.

Techniques: Mouse Assay, Staining, TUNEL Assay, Positive Control, Incubation, Negative Control, Labeling

Journal:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

doi: 10.1016/j.jmb.2007.03.006

Figure Lengend Snippet: Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

Techniques:

Journal:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

doi: 10.1016/j.jmb.2007.03.006

Figure Lengend Snippet: Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

Techniques:

Journal:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

doi: 10.1016/j.jmb.2007.03.006

Figure Lengend Snippet: DNase I protection on P/Ts terminated with dT analogues

Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

Techniques:

Journal:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

doi: 10.1016/j.jmb.2007.03.006

Figure Lengend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

Techniques:

Journal: Scientific Reports

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle

doi: 10.1038/s41598-018-25635-x

Figure Lengend Snippet: Perlecan deficiency does not affect cell turnover in white adipose tissue. ( a ) TUNEL staining in the VAT of the WT-Tg (upper panel) and the Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The areas containing positive nuclei of adipocytes are selected and shown. The positive control was made using DNase I. Note that TUNEL-positive nuclei are brown and negative nuclei are blue. ( b ) The percentage of TUNEL-positive nuclei of adipocytes. The nuclei from at least 100 adipocytes per mouse were evaluated. Data points and error bars represent the mean ± S.D. (n = 5). ( c ) Representative immunohistochemical staining of Ki67 in the VAT of the WT-Tg (upper panel) and the Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The tissue from human abdominal cancer was used as positive control. No nuclei were positive for Ki67 in any groups. Data were analyzed by two-way ANOVA with Tukey’s multiple comparison ( b ). Scale bar, 50  μm.

Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA.

Techniques: TUNEL Assay, Staining, Mouse Assay, Positive Control, Immunohistochemistry

Journal: Molecular microbiology

Article Title: The secretion ATPase ComGA is required for the binding and transport of transforming DNA

doi: 10.1111/j.1365-2958.2011.07730.x

Figure Lengend Snippet: Localization of DNAase-resistant DNA in the protoplasts of selected comGA mutants. Competent cells were incubated with a saturating amount of radiolabeled DNA. Following the addition of DNAase and centrifugal washing, samples were taken to determine the

Article Snippet: To determine DNA uptake 50 μg/ml of pancreatic DNAase (Sigma) was added to cell suspensions and incubated for 3 min at 37°C just prior to washing.

Techniques: Incubation

Journal: Microbiology

Article Title: Differential response of Porphyromonas gingivalis to varying levels and duration of hydrogen peroxide-induced oxidative stress

doi: 10.1099/mic.0.056416-0

Figure Lengend Snippet: (a) Functional distribution of P. gingivalis genes after 0.1, 0.25 and 0.5 mM H 2 O 2 treatment. DNase-treated total RNA extracted from P. gingivalis W83 after 0.1, 0.25 or 0.5 mM H 2 O 2 treatment was subjected to DNA microarray analysis. Functional gene classes

Article Snippet: Total RNA samples were obtained from P. gingivalis using the Ribopure RNA isolation kit (Ambion) and were subsequently treated with the DNase kit (Ambion) according to the manufacturer’s protocol. cDNA was synthesized from the RNA using the transcriptor high-fidelity cDNA synthesis kit (Roche).

Techniques: Functional Assay, Microarray

Journal: mBio

Article Title: A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA

doi: 10.1128/mBio.02805-18

Figure Lengend Snippet: C. heterostrophus secretes DNases, and secretion is induced by plant tissue. WT, nuc1 mutant, nuc2 mutant, and nuc1 nuc2 double-mutant filtrates degrade intact λ DNA in the presence of plant material. The nuc1 single mutant and nuc1 nuc2 double mutant degrade lambda DNA less well than the WT or the nuc2 single mutant. This indicates that DNase(s) are secreted by the fungus, that the nuc1 mutant secretes a DNase that is important in DNA degradation, and that secretion is induced by host material. Left, size markers in kilobases. +, addition of corn leaf (CL) fragments, lambda DNA, or purified RQ1 RNase-free DNase. Culture filtrates examined were from WT strain C4, nuc1 mutant strains 144206-3-1 and 8-1, nuc2 mutant strains 149183-2-1 and 3-1, and nuc1 nuc2 double-mutant strains 144206/149183-4-1 and 8-1. The negative-control reaction with λ DNA did not degrade the DNA, while the positive-control reaction with λ DNA plus DNase did. Also note that λ DNA was not degraded by the CL material.

Article Snippet: Reactions were run on 1% agarose gels at 100 V for 15 min. As a positive control for λ DNA degradation, 1 unit of RQ1 DNase (catalog no. M610A; Promega) was used.

Techniques: Mutagenesis, Lambda DNA Preparation, Purification, Negative Control, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: A Role for Huntington Disease Protein in Dendritic RNA Granules *

doi: 10.1074/jbc.M110.114561

Figure Lengend Snippet: Huntingtin co-localizes with dendritic mRNA in rodent neurons. A , mouse hippocampal neurons (DIV 14) stained live with SYTO RNASelect, subsequently incubated with DNase or RNase where indicated, and lastly probed with α-Htt and α-MAP2 antibodies. Scale bar , 5 μm. B , mouse hippocampal neurons were probed with α-Htt or α-Staufen antibodies and oligo(dT) FISH. Scale bar , 5 μm. C , Rat E19 cortical neurons (DIV 5) transfected with Htt480-17Q were stained with SYTO RNASelect 24 h after transfection. Htt and RNA are shown in red and green , respectively. Enlarged areas 1 and 2 are shown in the lower panel. Scale bar ( top , merge ), 30 μm. D , merged time-lapse images of RFP-Htt480-17Q and SYTO RNASelect. *, initial location of RFP-Htt480-17Q, and the arrow ). Scale bar , 1 μm.

Article Snippet: For the treated neurons (DNase & RNase), after SYTO® RNASelect incubation, the cells were permeabilized and incubated with PBS containing RQ1 RNase-Free DNase (2 μl of 1 unit/μl, Promega, Madison, WI) or RNase (1–2 μl of 10 mg/ml, Sigma) for 30 min at room temperature, then fixed with −20 °C methanol for 3 min.

Techniques: Staining, Incubation, Fluorescence In Situ Hybridization, Transfection