Structured Review

Millipore dna fragments
Acid and <t>DNA</t> damage resistance. A , relative survival of wild type and Δ <t>yybT</t> strain under acid stress conditions. B , spore formation efficiency for the wild type B. subtilis and Δ yybT mutant in the presence of DNA damage-inducing agent
Dna Fragments, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity *"

Article Title: YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity *

Journal:

doi: 10.1074/jbc.M109.040238

Acid and DNA damage resistance. A , relative survival of wild type and Δ yybT strain under acid stress conditions. B , spore formation efficiency for the wild type B. subtilis and Δ yybT mutant in the presence of DNA damage-inducing agent
Figure Legend Snippet: Acid and DNA damage resistance. A , relative survival of wild type and Δ yybT strain under acid stress conditions. B , spore formation efficiency for the wild type B. subtilis and Δ yybT mutant in the presence of DNA damage-inducing agent

Techniques Used: Mutagenesis

2) Product Images from "Cellular Growth and Mitochondrial Ultrastructure of Leishmania (Viannia) braziliensis Promastigotes Are Affected by the Iron Chelator 2,2-Dipyridyl"

Article Title: Cellular Growth and Mitochondrial Ultrastructure of Leishmania (Viannia) braziliensis Promastigotes Are Affected by the Iron Chelator 2,2-Dipyridyl

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002481

TUNEL assay. Promastigotes cultivated in control medium or treated with 100 µM 2,2-dipyridyl for 24 h (A) or 48 h (B) were examined by phase contrast microscopy and fluorescence microscopy at 100×. DAPI (blue channel) revealed intact nuclei and kinetoplasts in both control and treated promastigotes. TUNEL (green channel) detected DNA fragmentation exclusively in promastigotes treated with DNase. Merged images show the localization of the nuclei and kinetoplasts inside the cells.
Figure Legend Snippet: TUNEL assay. Promastigotes cultivated in control medium or treated with 100 µM 2,2-dipyridyl for 24 h (A) or 48 h (B) were examined by phase contrast microscopy and fluorescence microscopy at 100×. DAPI (blue channel) revealed intact nuclei and kinetoplasts in both control and treated promastigotes. TUNEL (green channel) detected DNA fragmentation exclusively in promastigotes treated with DNase. Merged images show the localization of the nuclei and kinetoplasts inside the cells.

Techniques Used: TUNEL Assay, Microscopy, Fluorescence

3) Product Images from "Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells"

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

Journal: bioRxiv

doi: 10.1101/2020.01.22.915934

BMP4 reduces global H3K4me3 promoter occupancy through reduction of its methyltransferases WDR82 and hSETD1A. A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p
Figure Legend Snippet: BMP4 reduces global H3K4me3 promoter occupancy through reduction of its methyltransferases WDR82 and hSETD1A. A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p

Techniques Used: Western Blot, Expressing, shRNA, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Related Articles

Isolation:

Article Title: Deletion of the Gene Encoding p60 in Listeria monocytogenes Leads to Abnormal Cell Division and Loss of Actin-Based Motility
Article Snippet: Restriction enzymes (Amersham Pharmacia Biotech), calf alkaline phosphatase (Amersham Pharmacia Biotech), and T4 DNA ligase (Life Technologies) were utilized according to the manufacturer's instructions. .. Chromosomal DNA from L. monocytogenes was isolated by resuspending bacteria in Tris-EDTA (pH 8.2) containing 2 mg of lysozyme (Sigma) ml−1 . .. After incubation at 37°C for 15 min, bacteria were harvested by centrifugation and further treated with DNAzol (Life Technologies) according to the manufacturer's instructions.

Fluorescence:

Article Title: In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response
Article Snippet: The GAG content was measured using the dimethylmethylene blue dye binding assay and the absorbance was measured at 525 nm using a microplate reader (Thermo, Karlsruhe, Germany). .. The cellularity was measured based on the DNA content using Hoechst 33258 (Sigma) and the fluorescence intensity was measured at 460 nm using a microplate fluorescence reader (FLX800, Bio-tec, Burlington, Vermont, USA). ..

Transfection:

Article Title: Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer
Article Snippet: For the HRR/ NHEJ balance, the ratio between green and red cells in each condition was calculated. .. Cell cycle DNA content analysis 48h following siRNA transfection, HeyA8 and IGROV1 cells were collected, fixed in ice-cold EtOH over-night and stained the following day with Propidium Iodide (PI, Sigma P4864). .. Analysis was performed on the Gallios flow cytometer and data were elaborated using Kaluza software (Beckman Coulter).

Staining:

Article Title: Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer
Article Snippet: For the HRR/ NHEJ balance, the ratio between green and red cells in each condition was calculated. .. Cell cycle DNA content analysis 48h following siRNA transfection, HeyA8 and IGROV1 cells were collected, fixed in ice-cold EtOH over-night and stained the following day with Propidium Iodide (PI, Sigma P4864). .. Analysis was performed on the Gallios flow cytometer and data were elaborated using Kaluza software (Beckman Coulter).

Article Title: The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus
Article Snippet: Primary antibodies were added to samples diluted in blocking buffer and visualized by second antibodies conjugated to Alexa 488 (green) or Alexa 594 (red) purchased from Molecular Probes (Leiden, The Netherlands). .. Viral and cellular DNA was stained with DAPI (4′-6-diamidino-2-phenylindole) purchased from Sigma, St. Louis, Mo. .. Cells were mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, Ala.), and in most studies, the cells were viewed at ×60/1.4 NA with a Nikon E800 microscope.

Article Title: Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro
Article Snippet: For wide-field immunofluorescence microscopy, cells on glass cover slips were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X100. .. After blocking and antibody staining in 0.5% BSA/0.2% gelatin in PBS (secondary antibody AlexaFluor 488-labeled, Invitrogen), nuclear DNA was stained by DAPI (Sigma Aldrich). .. Gene expression analysis Total RNA was extracted from live cells with Trizol reagent (Invitrogen).

Polymerase Chain Reaction:

Article Title: Role of toll-like receptor signalling in Aβ uptake and clearance
Article Snippet: .. Mouse tail DNA was extracted using the Extract-N-Amp tissue PCR kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s protocol. .. The PCR was carried out in 25 μl vol using 1 μl of tail DNA extract and 200 nM each of two primers for the TLR4 gene through 35 cycles consisting of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C.

Blocking Assay:

Article Title: Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro
Article Snippet: For wide-field immunofluorescence microscopy, cells on glass cover slips were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X100. .. After blocking and antibody staining in 0.5% BSA/0.2% gelatin in PBS (secondary antibody AlexaFluor 488-labeled, Invitrogen), nuclear DNA was stained by DAPI (Sigma Aldrich). .. Gene expression analysis Total RNA was extracted from live cells with Trizol reagent (Invitrogen).

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    Millipore cpg dna fragments
    Presence of DNase I hypersensitive sites within <t>CpG</t> islands 32 and 27 in the absence of <t>DNA</t> 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.
    Cpg Dna Fragments, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fragmented apoptotic dna
    ( 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 <t>apoptotic</t> 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 <t>DNA</t> (% 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
    Fragmented Apoptotic Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore genomic dna fragments encoding prkg2 exon iii
    NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for <t>prkg2</t> 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 <t>DNA</t> 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
    Genomic Dna Fragments Encoding Prkg2 Exon Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna fragmentation
    LMP2A or genistein inhibits BCR-induced tyrosine phosphorylation, <t>DNA</t> fragmentation, and cleavage <t>PARP</t> in Ramos cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) Ramos cells (1 × 10 6 /ml) were treated without (control [−])
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    Image Search Results


    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.

    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

    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.

    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

    ( 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

    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

    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

    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

    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

    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

    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

    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

    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 [−])

    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

    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.

    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:

    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

    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