dna fragments  (Millipore)

Journal: The Journal of Experimental Medicine

Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

doi: 10.1084/jem.20100204

Figure Lengend Snippet: Presence of DNase I hypersensitive sites within CpG islands 32 and 27 in the absence of DNA methylation. (A) The diagram depicts the approximate location of the genomic features of the BCL6 locus along with the restriction fragments and probes used for indirect end labeling. The approximate locations of the observed hypersensitive sites are indicated on the diagram. (B) The 5′ end of the BCL6 locus was analyzed by DNase I digestion using the reference restriction enzyme XbaI. Wedges indicate concentrations of DNase I. The locations of restriction sites and the probe used for the Southern blot are depicted in A. Arrows indicate the major DNase I hypersensitive sites and their locations are summarized in A. Marker positions were measured from ethidium-stained gel before transfer. Data shown is representative of results from at least three independent experiments.

Article Snippet: Input and methylated CpG DNA fragments were amplified (WGA2; Sigma-Aldrich), labeled with Cy3 and Cy5 random monomers (TriLink Biotechnologies), respectively, and hybridized onto a NimbleGen 2.1M Deluxe Human Promoter Array (Roche).

Techniques: DNA Methylation Assay, End Labeling, Southern Blot, Marker, Staining

Journal: The Journal of Experimental Medicine

Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

doi: 10.1084/jem.20100204

Figure Lengend Snippet: Cell type–specific DNA methylation status at BCL6 intronic region. (A) The diagram depicts the genomic organization of the human BCL6 locus. Indicated are the two transcription start sites (arrows), the four CpG islands (17, 32, 27, and 38), and the exons (black rectangles). Below the cartoon, the results of genomic bisulfite sequencing are presented. Each line of circles indicates an individual clone sequenced in the analysis after bisulfite treatment and PCR. Open circles indicate CpG sites at which no DNA methylation is detected. Blackened circles indicate CpG sites which are methylated. Data shown is representative of the results of three independent biological replicates. (B) MIRA-chip analysis of Raji (top) and H929 (bottom) across the 5′ end of the BCL6 locus. Each vertical line represents the mean normalized log 2 ratio of enriched/input probe signal from two replicate samples, corresponding to the genomic location on chromosome 3 at the BCL6 locus (UCSC genome browser HG18) as listed on the x axis. Blue and yellow colors represent methylated and unmethylated regions, respectively.

Article Snippet: Input and methylated CpG DNA fragments were amplified (WGA2; Sigma-Aldrich), labeled with Cy3 and Cy5 random monomers (TriLink Biotechnologies), respectively, and hybridized onto a NimbleGen 2.1M Deluxe Human Promoter Array (Roche).

Techniques: DNA Methylation Assay, Methylation Sequencing, Polymerase Chain Reaction, Methylation, Chromatin Immunoprecipitation

Journal: Journal of Cell Death

Article Title: Increased Lysosomal Membrane Permeabilization in Oxidant-exposed Macrophages of Human Fibrotic Lungs

doi: 10.4137/JCD.S13271

Figure Lengend Snippet: ( A ) Apoptosis, necrosis, and total cell death in cultures of oxidatively stressed human lung macrophages retrieved from healthy subjects (n = 7) and patients with lung fibrosis (n = 7). Cell death (apoptosis and necrosis), which appeared 10 h after the end of oxidant challenge in cultures of lung macrophages from healthy subjects ( B ) and patients with lung fibrosis ( C ) was assessed by typical morphology using a phase contrast microscope. The fraction of apoptotic cells at 10 h post-oxidative stress in cultures of lung macrophages from healthy subjects ( D and F ) and patients with lung fibrosis ( E and G ) was also assessed using Giemsa staining ( D and E ) and by the fraction of apoptotic sub-G 1 DNA (% of total is indicated; F and G ). All methods gave similar results for apoptosis. Representative micrographs and stack bars of oxidant-challenged cultures of lung macrophages are shown. Representative apoptotic cells are indicated with black arrows. Detached cells were all necrotic. In the non-oxidatively stressed cultures, cell death was

Article Snippet: – Fragmented apoptotic DNA, stained with propidium iodide (Sigma), was assessed using cytofluorometry.

Techniques: Microscopy, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

doi: 10.1002/jbmr.2909

Figure Lengend Snippet: NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p

Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

Techniques: Isolation, Mouse Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Cell Culture, ALP Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

doi: 10.1002/jbmr.2909

Figure Lengend Snippet: NO-Cbi inhibits osteoclast differentiation (A,B) Murine bone marrow mono-nuclear cells were cultured in the presence of M-CSF; after 3 d, RANKL was added, with or without NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μM NO-Cbi, 5 μM DETA-NONOate (Deta-NO, which releases two moles of NO per mole of drug), or 100 μM 8-pCPT-cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. (E–G) Bone marrow mononuclear cells isolated from PRKG2 f/f mice were cultured with M-CSF and RANKL as described in panel A, but 24 h after plating, cells were infected with adenovirus expressing either green fluorescent protein (GFP, control) or CRE. Relative amounts of prkg2 mRNA were determined by qRT-PCR (E), and TRAP-positive cells were counted on day 8 (F,G). Panels B–F show means ± SEM of at least three independent experiments; *p

Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Infection

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

doi: 10.1002/jbmr.2909

Figure Lengend Snippet: NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p

Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

Techniques: Expressing, Isolation, Mouse Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Cell Culture, ALP Assay, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Epstein-Barr Virus (EBV) Latent Membrane Protein 2A Regulates B-Cell Receptor-Induced Apoptosis and EBV Reactivation through Tyrosine Phosphorylation

doi: 10.1128/JVI.79.13.8655-8660.2005

Figure Lengend Snippet: LMP2A or genistein inhibits BCR-induced tyrosine phosphorylation, DNA fragmentation, and cleavage PARP in Ramos cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) Ramos cells (1 × 10 6 /ml) were treated without (control [−])

Article Snippet: To verify the DNA fragmentation and cleavage of PARP by BCR ligation were dependent on caspase activity in Ramos cells, cells were pretreated with zVAD-fmk (Calbiochem, La Jolla, CA), a broad caspase inhibitor and, as expected, DNA fragmentation and PARP cleavage were blocked by the addition of zVAD-fmk (Fig. and ).

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Virology

Article Title: Epstein-Barr Virus (EBV) Latent Membrane Protein 2A Regulates B-Cell Receptor-Induced Apoptosis and EBV Reactivation through Tyrosine Phosphorylation

doi: 10.1128/JVI.79.13.8655-8660.2005

Figure Lengend Snippet: LMP2A inhibits BCR-induced DNA fragmentation and cleavage of PARP in Ramos cells. (A) Cells were seeded at 3 × 10 5 cells/ml, and cells were treated without (control) or with 35 μg/ml anti-IgM antibody (αIgAb) for 24 h or 48 h.

Article Snippet: To verify the DNA fragmentation and cleavage of PARP by BCR ligation were dependent on caspase activity in Ramos cells, cells were pretreated with zVAD-fmk (Calbiochem, La Jolla, CA), a broad caspase inhibitor and, as expected, DNA fragmentation and PARP cleavage were blocked by the addition of zVAD-fmk (Fig. and ).

Techniques:

Journal: Journal of Virology

Article Title: Epstein-Barr Virus (EBV) Latent Membrane Protein 2A Regulates B-Cell Receptor-Induced Apoptosis and EBV Reactivation through Tyrosine Phosphorylation

doi: 10.1128/JVI.79.13.8655-8660.2005

Figure Lengend Snippet: LMP2A or genistein blocks BCR-induced DNA fragmentation, cleavage of PARP, and EBV reactivation. (A) DNA fragmentation and evaluation of cleavage of PARP. Parental (P), vector control (V), and LMP2A-expressing (2A) Akata cells (5 × 10 5 /ml) were

Article Snippet: To verify the DNA fragmentation and cleavage of PARP by BCR ligation were dependent on caspase activity in Ramos cells, cells were pretreated with zVAD-fmk (Calbiochem, La Jolla, CA), a broad caspase inhibitor and, as expected, DNA fragmentation and PARP cleavage were blocked by the addition of zVAD-fmk (Fig. and ).

Techniques: Plasmid Preparation, Expressing