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    Structured Review

    Thermo Fisher prkdc dna pkcs
    <t>DNA</t> repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors <t>DNA-PKcs</t> <t>(Prkdc),</t> DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p
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    Images

    1) Product Images from "DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks"

    Article Title: DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01312

    DNA repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors DNA-PKcs (Prkdc), DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p
    Figure Legend Snippet: DNA repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors DNA-PKcs (Prkdc), DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p

    Techniques Used: Expressing, Western Blot, Cell Culture, Irradiation

    DN2 thymocytes activate a robust DNA damage response following γ-irradiation. Western blot analysis of (A) H2AX (Ser139) phosphorylation (γH2AX—marker of DNA double-strand breaks), p53 stabilization; and p21 and Puma expression in DN2 cells and NH-hematopoietic stem cells (HSCs) at 0–24 h post 4 Gy irradiation. Western blot analysis of total endogenous levels of (B) of H2AX, ATM, DNA-PKcs and Chk2; (C) of 53BP1, DNA ligase IV and Rad51; and (D) of Bcl-2, Bcl-XL, and Bim in un-irradiated (control) DN2 cells and NH-HSCs. β-Actin and β-tubulin were used as internal controls. All images are representative of one of three independent experiments.
    Figure Legend Snippet: DN2 thymocytes activate a robust DNA damage response following γ-irradiation. Western blot analysis of (A) H2AX (Ser139) phosphorylation (γH2AX—marker of DNA double-strand breaks), p53 stabilization; and p21 and Puma expression in DN2 cells and NH-hematopoietic stem cells (HSCs) at 0–24 h post 4 Gy irradiation. Western blot analysis of total endogenous levels of (B) of H2AX, ATM, DNA-PKcs and Chk2; (C) of 53BP1, DNA ligase IV and Rad51; and (D) of Bcl-2, Bcl-XL, and Bim in un-irradiated (control) DN2 cells and NH-HSCs. β-Actin and β-tubulin were used as internal controls. All images are representative of one of three independent experiments.

    Techniques Used: Irradiation, Western Blot, Marker, Expressing

    2) Product Images from "The Vacuolar Protein Sorting-38 Subunit of the Arabidopsis Phosphatidylinositol-3-Kinase Complex Plays Critical Roles in Autophagy, Endosome Sorting, and Gravitropism"

    Article Title: The Vacuolar Protein Sorting-38 Subunit of the Arabidopsis Phosphatidylinositol-3-Kinase Complex Plays Critical Roles in Autophagy, Endosome Sorting, and Gravitropism

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00781

    Arabidopsis VPS38 interacts with other subunits of class-III PtdIns-3 kinase complex. (A) Pairwise Y2H assays using the GAL4-based system showing that VPS38 interacts with ATG6. VSPS38 fused to the N terminus of the DNA-binding domain (BD) and ATG6 fused to the N terminus of the activation domain (AD) were co-expressed in yeast and tested for binding by growth on synthetic complete medium lacking leucine, tryptophan, and histidine (-L-W-H), and containing 3-amino-1,2,4-triazole (+3AT). Viability of the cells was confirmed by growth on medium lacking leucine and tryptophan (-L-W). (B) Pairwise Y2H assays by the split-ubiquitin mating system showing interactions among VPS38, ATG6, VPS15, and VPS34. Each full-length protein was expressed as a fusion to either Cub-PLV as bait or NubG as prey, and co-expressed in diploid yeast cells. Positive interactions were determined by growth of twofold serial dilutions on synthetic complete medium lacking uracil, methionine, leucine, tryptophan, histidine, and adenine (-Ura-M-L-W-H-Ade). The empty NubG and NubWT vectors were used as negative and positive controls, respectively. Viability of the cells was confirmed by growth on synthetic complete medium lacking uracil, methionine, leucine, and tryptophan (-Ura-M-L-W). (C) Pairwise BiFC assays showing the interactions among VPS38, ATG6, VPS15, and VPS34 in planta . Each full-length protein was expressed as a fusion to either N-terminal fragment (nYFP) or C-terminal fragment (cYFP) of YFP and then transiently co-expressed in N. benthamiana leaf epidermal cells. Appearance of the fluorescent signals was observed by confocal microscopic analysis 36 h after infiltration. Scale bar = 20 μm. (D) Schematic of the interactions detected among VPS38, ATG6, VPS15, and VPS34. The arrow thickness is an estimate of binding strength based on all interaction assays. The solid and dashed lines indicate interactions that were demonstrated by both Y2H and BiFC, or by just one of the methods, respectively.
    Figure Legend Snippet: Arabidopsis VPS38 interacts with other subunits of class-III PtdIns-3 kinase complex. (A) Pairwise Y2H assays using the GAL4-based system showing that VPS38 interacts with ATG6. VSPS38 fused to the N terminus of the DNA-binding domain (BD) and ATG6 fused to the N terminus of the activation domain (AD) were co-expressed in yeast and tested for binding by growth on synthetic complete medium lacking leucine, tryptophan, and histidine (-L-W-H), and containing 3-amino-1,2,4-triazole (+3AT). Viability of the cells was confirmed by growth on medium lacking leucine and tryptophan (-L-W). (B) Pairwise Y2H assays by the split-ubiquitin mating system showing interactions among VPS38, ATG6, VPS15, and VPS34. Each full-length protein was expressed as a fusion to either Cub-PLV as bait or NubG as prey, and co-expressed in diploid yeast cells. Positive interactions were determined by growth of twofold serial dilutions on synthetic complete medium lacking uracil, methionine, leucine, tryptophan, histidine, and adenine (-Ura-M-L-W-H-Ade). The empty NubG and NubWT vectors were used as negative and positive controls, respectively. Viability of the cells was confirmed by growth on synthetic complete medium lacking uracil, methionine, leucine, and tryptophan (-Ura-M-L-W). (C) Pairwise BiFC assays showing the interactions among VPS38, ATG6, VPS15, and VPS34 in planta . Each full-length protein was expressed as a fusion to either N-terminal fragment (nYFP) or C-terminal fragment (cYFP) of YFP and then transiently co-expressed in N. benthamiana leaf epidermal cells. Appearance of the fluorescent signals was observed by confocal microscopic analysis 36 h after infiltration. Scale bar = 20 μm. (D) Schematic of the interactions detected among VPS38, ATG6, VPS15, and VPS34. The arrow thickness is an estimate of binding strength based on all interaction assays. The solid and dashed lines indicate interactions that were demonstrated by both Y2H and BiFC, or by just one of the methods, respectively.

    Techniques Used: Binding Assay, Activation Assay, Bimolecular Fluorescence Complementation Assay

    3) Product Images from "Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis"

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007117

    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Figure Legend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Techniques Used: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Figure Legend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Techniques Used: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR
    Figure Legend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Techniques Used: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR
    Figure Legend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Techniques Used: Mass Spectrometry, Cell Culture

    Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.
    Figure Legend Snippet: Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Techniques Used: Binding Assay, Recombinant, De-Phosphorylation Assay, Infection

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).
    Figure Legend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Techniques Used: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    CA and p12 co-localise on mitotic chromatin independently of BET-protein binding. HeLa cells synchronised using a double-aphidicolin block were infected with WT Mo-MLV VLPs or mutants carrying p12_mut14 or IN_W390A. 10 h post-infection, the cells were fixed, stained for p12 (anti-p12, red), CA (anti-CA, green) and DNA (DAPI, blue), and analysed by confocal microscopy. (A) Representative images showing WT p12 and CA co-localisation on mitotic chromatin. Bottom panels are enlarged views of boxed regions in top panels. (B) Representative images of cells infected with VLPs carrying p12_WT (top panels) or p12_mut14 (bottom panels). (C) A representative image of cells infected with VLPs carrying IN_W390A, which is deficient in BET protein binding. (D) Infectivity of Mo-MLV VLPs carrying alterations in p12 and/or IN. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying p12_WT, p12_mut14, p12+ h CBS or (p12+ h CBS)_mut14 in combination with IN_WT or BET-binding deficient IN_W390A. Infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).
    Figure Legend Snippet: CA and p12 co-localise on mitotic chromatin independently of BET-protein binding. HeLa cells synchronised using a double-aphidicolin block were infected with WT Mo-MLV VLPs or mutants carrying p12_mut14 or IN_W390A. 10 h post-infection, the cells were fixed, stained for p12 (anti-p12, red), CA (anti-CA, green) and DNA (DAPI, blue), and analysed by confocal microscopy. (A) Representative images showing WT p12 and CA co-localisation on mitotic chromatin. Bottom panels are enlarged views of boxed regions in top panels. (B) Representative images of cells infected with VLPs carrying p12_WT (top panels) or p12_mut14 (bottom panels). (C) A representative image of cells infected with VLPs carrying IN_W390A, which is deficient in BET protein binding. (D) Infectivity of Mo-MLV VLPs carrying alterations in p12 and/or IN. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying p12_WT, p12_mut14, p12+ h CBS or (p12+ h CBS)_mut14 in combination with IN_WT or BET-binding deficient IN_W390A. Infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Techniques Used: Protein Binding, Blocking Assay, Infection, Staining, Confocal Microscopy, Binding Assay, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.
    Figure Legend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Techniques Used: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging

    4) Product Images from "The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1"

    Article Title: The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.00244

    CagRS regulates the biosynthesis of arginine-the direct precursor of CA. (A) Schematic diagram of the arginine biosynthetic gene cluster. P1–P5 are different promoter regions of the arginine gene cluster: P1, 211 bp of the argH upstream region; P2, 299 bp of the argG upstream region; P3, 154 bp of the argB - D - R upstream region; P4, 271 bp of the argJ upstream region; P5, 160 bp of the argC upstream region. (B) The binding of CagR to the promoter regions of argC and argG. EMSAs of argG, argB, argC, argJ , and argH with purified His-tagged CagR. The promoter fragments were labeled with biotin-11-UTP using the Biotin 3′ End DNA Labeling kit. The above probes were incubated either with no protein (–) or 3.0 μg CagR (+). The appropriate amount of polydI/dC (1.0 mg) was used as competitor. (C) Comparison of argG and argC promoter sequences with the conserved CGCNGCCG motif for CagR binding.
    Figure Legend Snippet: CagRS regulates the biosynthesis of arginine-the direct precursor of CA. (A) Schematic diagram of the arginine biosynthetic gene cluster. P1–P5 are different promoter regions of the arginine gene cluster: P1, 211 bp of the argH upstream region; P2, 299 bp of the argG upstream region; P3, 154 bp of the argB - D - R upstream region; P4, 271 bp of the argJ upstream region; P5, 160 bp of the argC upstream region. (B) The binding of CagR to the promoter regions of argC and argG. EMSAs of argG, argB, argC, argJ , and argH with purified His-tagged CagR. The promoter fragments were labeled with biotin-11-UTP using the Biotin 3′ End DNA Labeling kit. The above probes were incubated either with no protein (–) or 3.0 μg CagR (+). The appropriate amount of polydI/dC (1.0 mg) was used as competitor. (C) Comparison of argG and argC promoter sequences with the conserved CGCNGCCG motif for CagR binding.

    Techniques Used: Binding Assay, Purification, Labeling, DNA Labeling, Incubation

    5) Product Images from "Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain"

    Article Title: Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain

    Journal: Cell

    doi: 10.1016/j.cell.2011.03.022

    5hmC-containing DNA Are Demethylated in HEK293 Cells (A) A schematic diagram of a PCR-based approach to generate 5mC-, or 5hmC-containing DNA fragments, that contain an Ubiquitin promoter (pUbC) followed by the GFP open reading frame. (B) Summary of CpG methylation levels of GFP TSS regions under different conditions as quantified by the HpaII sensitivity. Open circles represent data from individual experiments and lines represent mean values (**: P
    Figure Legend Snippet: 5hmC-containing DNA Are Demethylated in HEK293 Cells (A) A schematic diagram of a PCR-based approach to generate 5mC-, or 5hmC-containing DNA fragments, that contain an Ubiquitin promoter (pUbC) followed by the GFP open reading frame. (B) Summary of CpG methylation levels of GFP TSS regions under different conditions as quantified by the HpaII sensitivity. Open circles represent data from individual experiments and lines represent mean values (**: P

    Techniques Used: Polymerase Chain Reaction, CpG Methylation Assay

    BER Enzyme Activities Are Required for 5hmC Demethylation (A and B) Effects of PARP1 inhibitor ABT-888 (50 μM) and APE inhibitor CRT0044876 (50 μM) on demethylation of transfected 5hmC-GFP DNA fragments. Shown in (A) is a summary of HpaII sensitivity assay. Open circles represent data from individual experiments and lines represent mean values (*: P
    Figure Legend Snippet: BER Enzyme Activities Are Required for 5hmC Demethylation (A and B) Effects of PARP1 inhibitor ABT-888 (50 μM) and APE inhibitor CRT0044876 (50 μM) on demethylation of transfected 5hmC-GFP DNA fragments. Shown in (A) is a summary of HpaII sensitivity assay. Open circles represent data from individual experiments and lines represent mean values (*: P

    Techniques Used: Transfection, Sensitive Assay

    AID/APOBEC Deaminases Promote 5hmC Demethylation (A and B) Overexpression of AID results in a decrease in the abundance of 5hmCs in genomic DNA samples from HEK 293 cells as monitored by immunoblotting (A) and ELISA (B) using anti-5hmC antibodies. (C) Effects of AID on demethylation of 5hmC-GFP and 5mC-GFP DNA. Values represent mean ± SEM (n = 3; *: P
    Figure Legend Snippet: AID/APOBEC Deaminases Promote 5hmC Demethylation (A and B) Overexpression of AID results in a decrease in the abundance of 5hmCs in genomic DNA samples from HEK 293 cells as monitored by immunoblotting (A) and ELISA (B) using anti-5hmC antibodies. (C) Effects of AID on demethylation of 5hmC-GFP and 5mC-GFP DNA. Values represent mean ± SEM (n = 3; *: P

    Techniques Used: Over Expression, Enzyme-linked Immunosorbent Assay

    TET1 and AID Regulate Endogenous 5hmC Levels and Promote DNA Demethylation in the Adult Mouse Brain (A and B) Effects of AAV-mediated overexpression of control YFP, TET1, TET1m (A), and AID (B) on the endogenous 5hmC levels as measured by immunoblotting (left) and ELISA (right). Engineered AAV viruses were stereotaxically injected into the dentate gyrus of adult mice and dentate gyrus tissue was micro-dissected one week later for analysis. Values represent mean ± SEM (n = 3 animals for each condition; **: P
    Figure Legend Snippet: TET1 and AID Regulate Endogenous 5hmC Levels and Promote DNA Demethylation in the Adult Mouse Brain (A and B) Effects of AAV-mediated overexpression of control YFP, TET1, TET1m (A), and AID (B) on the endogenous 5hmC levels as measured by immunoblotting (left) and ELISA (right). Engineered AAV viruses were stereotaxically injected into the dentate gyrus of adult mice and dentate gyrus tissue was micro-dissected one week later for analysis. Values represent mean ± SEM (n = 3 animals for each condition; **: P

    Techniques Used: Over Expression, Enzyme-linked Immunosorbent Assay, Injection, Mouse Assay

    6) Product Images from "Tilted post arrays for separating long DNA"

    Article Title: Tilted post arrays for separating long DNA

    Journal: Biomicrofluidics

    doi: 10.1063/1.4884521

    Average velocity and the variance of the peak width for the 20 kbp DNA and 48.5 kbp DNA as a function of electric field strength. The error bars correspond to the standard deviation of the data set. The dashed lines are included to guide the eye.
    Figure Legend Snippet: Average velocity and the variance of the peak width for the 20 kbp DNA and 48.5 kbp DNA as a function of electric field strength. The error bars correspond to the standard deviation of the data set. The dashed lines are included to guide the eye.

    Techniques Used: Standard Deviation

    Representative electropherograms for 10 V/cm, 30 V/cm, and 50 V/cm for 20 kbp and 48.5 kbp DNA with finish line detection at 4 mm inside the array. The peak identities are labeled only for the 30 V/cm electropherogram. The inset presents the resolution as a function of electric field obtained over multiple devices and multiple days.
    Figure Legend Snippet: Representative electropherograms for 10 V/cm, 30 V/cm, and 50 V/cm for 20 kbp and 48.5 kbp DNA with finish line detection at 4 mm inside the array. The peak identities are labeled only for the 30 V/cm electropherogram. The inset presents the resolution as a function of electric field obtained over multiple devices and multiple days.

    Techniques Used: Labeling

    7) Product Images from "Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme"

    Article Title: Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1368

    DNA recognition by BfiI-C and EcoRII-N. ( A ) The view of the BfiI-C–DNA complex along the long DNA axis (left) and the side view (right). The DNA-recognition site is colored dark grey. The secondary structure elements of the N-arm (α-helix α6, β-strand β11) and the C-arm (β-strands β14 and β15) are colored green and orange, respectively. Spheres of the matching colors represent the Cα atoms of the DNA-recognition residues from the N- and C-arms. The additional DNA-recognition element N-loop (residues 245–252) is colored blue and the C-loop (residues 335–341) is red. A region of the top DNA strand (nucleotides A4-G7) and adjacent recognition residues are shown against their mF O -DF C SIGMAA-weighted-electron density contoured at 2.0 σ level. ( B ) The sequence and numbering of the cognate 12/12 oligoduplex used in this study. DNA bases that interact with the N- and C-arms are boxed in green and orange, respectively. ( C ) Recognition of individual base pairs by BfiI-C. Panels for the individual base pairs are arranged following the top strand ACTGGG in the 5′→3′ direction. The N- and C-arm residue labels are colored as in panel (A). ( D and E ) The sequence and numbering of the cognate EcoRII-N oligoduplex and the recognition of individual base pairs by EcoRII-N [PDB ID 3HQF ( 7 )]. The residue labels and boxes encircling EcoRII-N sequence elements are colored as in panels (B and C).
    Figure Legend Snippet: DNA recognition by BfiI-C and EcoRII-N. ( A ) The view of the BfiI-C–DNA complex along the long DNA axis (left) and the side view (right). The DNA-recognition site is colored dark grey. The secondary structure elements of the N-arm (α-helix α6, β-strand β11) and the C-arm (β-strands β14 and β15) are colored green and orange, respectively. Spheres of the matching colors represent the Cα atoms of the DNA-recognition residues from the N- and C-arms. The additional DNA-recognition element N-loop (residues 245–252) is colored blue and the C-loop (residues 335–341) is red. A region of the top DNA strand (nucleotides A4-G7) and adjacent recognition residues are shown against their mF O -DF C SIGMAA-weighted-electron density contoured at 2.0 σ level. ( B ) The sequence and numbering of the cognate 12/12 oligoduplex used in this study. DNA bases that interact with the N- and C-arms are boxed in green and orange, respectively. ( C ) Recognition of individual base pairs by BfiI-C. Panels for the individual base pairs are arranged following the top strand ACTGGG in the 5′→3′ direction. The N- and C-arm residue labels are colored as in panel (A). ( D and E ) The sequence and numbering of the cognate EcoRII-N oligoduplex and the recognition of individual base pairs by EcoRII-N [PDB ID 3HQF ( 7 )]. The residue labels and boxes encircling EcoRII-N sequence elements are colored as in panels (B and C).

    Techniques Used: Sequencing

    Recognition of the 3′-terminal nucleotides by BfiI-C and EcoRII-N. The C-arms of BfiI-C and EcoRII-N are orange and pink, respectively, the top DNA strand is white, the bottom DNA strand is grey. Only the last 3 bp of the BfiI recognition site and the overlapping base pairs from the EcoRII-N–DNA structure (PDB ID 3HQF) are shown.
    Figure Legend Snippet: Recognition of the 3′-terminal nucleotides by BfiI-C and EcoRII-N. The C-arms of BfiI-C and EcoRII-N are orange and pink, respectively, the top DNA strand is white, the bottom DNA strand is grey. Only the last 3 bp of the BfiI recognition site and the overlapping base pairs from the EcoRII-N–DNA structure (PDB ID 3HQF) are shown.

    Techniques Used:

    B3 and B3-like DBDs of the pseudobarrel fold (SCOP number 101935). ( A ) Apo DBD (BfiI-C) of the BfiI restriction enzyme [residues 193–358 of B chain, PDB ID 2C1L ( 4 )]. ( B ) The effector domain of the EcoRII restriction enzyme (EcoRII-N) in the DNA-bound form [PDB ID 3HQF, ( 7 )]. ( C ) The NMR structure of the RAV1-B3 domain [model 1 in PDB ID 1WID ( 5 )]. Common structural core made of 7 β-strands is colored in magenta, DNA backbone in (B) is depicted as a black double-helix.
    Figure Legend Snippet: B3 and B3-like DBDs of the pseudobarrel fold (SCOP number 101935). ( A ) Apo DBD (BfiI-C) of the BfiI restriction enzyme [residues 193–358 of B chain, PDB ID 2C1L ( 4 )]. ( B ) The effector domain of the EcoRII restriction enzyme (EcoRII-N) in the DNA-bound form [PDB ID 3HQF, ( 7 )]. ( C ) The NMR structure of the RAV1-B3 domain [model 1 in PDB ID 1WID ( 5 )]. Common structural core made of 7 β-strands is colored in magenta, DNA backbone in (B) is depicted as a black double-helix.

    Techniques Used: Nuclear Magnetic Resonance

    Sequence and structure elements involved in protein–DNA interactions in B3-like domains. ( A ) Structure-based multiple sequence alignment of BfiI-C (PDB ID 2C1L, chain A), EcoRII-N (PDB ID 3HQF), RAV1-B3 (PDB ID 1WID, model 1), VRN1-B3 (PDB ID 4I1K, chain A) and At1g16640-B3 (PDB ID 1YEL, model 1) was generated by MultiProt and Staccato ( 31 ). Residues and secondary structure elements are numbered according to the BfiI-C–DNA structure. BfiI-C DNA-binding elements: N-arm, N-loop, C-arm and C-loop are marked by green, blue, orange and red stripes, respectively. The (D/E)XR motif of BfiI-C and EcoRII-N responsible for recognition of the 5′-TGG-3′ trinucleotide is marked by black boxes and black circles. Residues contacting the ‘clamp’ phosphates are marked by magenta boxes and asterisks. The figure was generated using ESPRIPT ( 32 ). ( B ) Interaction of B3-like domains with DNA: BfiI-C (this study), EcoRII-N [PDB ID 3HQF, ( 7 )] and RAV1-B3 [PDB ID 1WID, interactions with DNA according to Yamasaki et al. ( 5 , 8 )]. The bases in the recognition sites are colored yellow; orange, green, blue and red islands encircle residues from the N-arm, C-arm, N-loop and C-loop, respectively. Residues contacting DNA phosphate oxygen atoms are depicted in the proximity of the corresponding phosphates. ‘Clamp’ phosphates are colored in cyan.
    Figure Legend Snippet: Sequence and structure elements involved in protein–DNA interactions in B3-like domains. ( A ) Structure-based multiple sequence alignment of BfiI-C (PDB ID 2C1L, chain A), EcoRII-N (PDB ID 3HQF), RAV1-B3 (PDB ID 1WID, model 1), VRN1-B3 (PDB ID 4I1K, chain A) and At1g16640-B3 (PDB ID 1YEL, model 1) was generated by MultiProt and Staccato ( 31 ). Residues and secondary structure elements are numbered according to the BfiI-C–DNA structure. BfiI-C DNA-binding elements: N-arm, N-loop, C-arm and C-loop are marked by green, blue, orange and red stripes, respectively. The (D/E)XR motif of BfiI-C and EcoRII-N responsible for recognition of the 5′-TGG-3′ trinucleotide is marked by black boxes and black circles. Residues contacting the ‘clamp’ phosphates are marked by magenta boxes and asterisks. The figure was generated using ESPRIPT ( 32 ). ( B ) Interaction of B3-like domains with DNA: BfiI-C (this study), EcoRII-N [PDB ID 3HQF, ( 7 )] and RAV1-B3 [PDB ID 1WID, interactions with DNA according to Yamasaki et al. ( 5 , 8 )]. The bases in the recognition sites are colored yellow; orange, green, blue and red islands encircle residues from the N-arm, C-arm, N-loop and C-loop, respectively. Residues contacting DNA phosphate oxygen atoms are depicted in the proximity of the corresponding phosphates. ‘Clamp’ phosphates are colored in cyan.

    Techniques Used: Sequencing, Generated, Binding Assay

    8) Product Images from "Transcriptome-wide discovery of circular RNAs in Archaea"

    Article Title: Transcriptome-wide discovery of circular RNAs in Archaea

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1009

    Identification of circular RNA products in RNA-seq data. ( A ) An RNA-seq cDNA read that maps to the reference DNA in a non-linear, chiastic manner, is a hallmark of circular RNA. ( B ) Schematic representation of the Sulfolobus solfataricus tRNA Trp , which contains a 65 b intron that is cleaved in the process of tRNA maturation and becomes a stable RNA circle. ( C ) Reads derived from the region around the circularization junction of the excised tRNA Trp intron show chiastic mapping to the genome of S. solfataricus P2, exemplifying the power of RNA-seq in circular RNA discovery. Multiple different reads spanning the circularization junction confirm that the observed circular junction is not an amplification artifact. Numbers denote position of the S. solfataricus P2 genome.
    Figure Legend Snippet: Identification of circular RNA products in RNA-seq data. ( A ) An RNA-seq cDNA read that maps to the reference DNA in a non-linear, chiastic manner, is a hallmark of circular RNA. ( B ) Schematic representation of the Sulfolobus solfataricus tRNA Trp , which contains a 65 b intron that is cleaved in the process of tRNA maturation and becomes a stable RNA circle. ( C ) Reads derived from the region around the circularization junction of the excised tRNA Trp intron show chiastic mapping to the genome of S. solfataricus P2, exemplifying the power of RNA-seq in circular RNA discovery. Multiple different reads spanning the circularization junction confirm that the observed circular junction is not an amplification artifact. Numbers denote position of the S. solfataricus P2 genome.

    Techniques Used: RNA Sequencing Assay, Derivative Assay, Amplification

    Experimental verification of cRNAs by RT-PCR. ( A ) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. ( B ) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. ( C ) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. ( D ) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, ( 1 ) ncRNA found in genomic location 1 275 500–1 275 567, ( 2 ) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.
    Figure Legend Snippet: Experimental verification of cRNAs by RT-PCR. ( A ) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. ( B ) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. ( C ) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. ( D ) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, ( 1 ) ncRNA found in genomic location 1 275 500–1 275 567, ( 2 ) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction, Northern Blot, Marker

    9) Product Images from "Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100"

    Article Title: Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100

    Journal: eLife

    doi: 10.7554/eLife.07295

    WRKY33 does not bind to the G/TTTGAAT motif. EMSA was performed using recombinant WRKY33 and biotin labeled DNA probes. A DNA oligonucleotide containing 3 W-box elements ( Mao et al., 2011 ) or a W-box mutated version (W-boxmut) hereof served as a positive and as a negative control, respectively ( A ). The 45 bp M-3 was derived from the PROPEP3 (At5g64905) gene promoter and contained three copies of the G/TTTGAAT motif, whereas the 40 bp M-7 was derived from the WAKL7 (At1g16090) gene promoter and contained one G/TTTGAAT motif. No binding of WRKY33 was observed to the W-boxmut probe and to both the M-3 and M-7 probes ( B ). Specificity of W-box binding was shown by competition assays using 250-fold (W-box) and 500-fold (W-box; M-3, M-7) excess of unlabeled probes. Protein lysates derived from IPTG-induced bacteria harboring the empty expression vector pMCSG48 served as an additional control. DOI: http://dx.doi.org/10.7554/eLife.07295.005
    Figure Legend Snippet: WRKY33 does not bind to the G/TTTGAAT motif. EMSA was performed using recombinant WRKY33 and biotin labeled DNA probes. A DNA oligonucleotide containing 3 W-box elements ( Mao et al., 2011 ) or a W-box mutated version (W-boxmut) hereof served as a positive and as a negative control, respectively ( A ). The 45 bp M-3 was derived from the PROPEP3 (At5g64905) gene promoter and contained three copies of the G/TTTGAAT motif, whereas the 40 bp M-7 was derived from the WAKL7 (At1g16090) gene promoter and contained one G/TTTGAAT motif. No binding of WRKY33 was observed to the W-boxmut probe and to both the M-3 and M-7 probes ( B ). Specificity of W-box binding was shown by competition assays using 250-fold (W-box) and 500-fold (W-box; M-3, M-7) excess of unlabeled probes. Protein lysates derived from IPTG-induced bacteria harboring the empty expression vector pMCSG48 served as an additional control. DOI: http://dx.doi.org/10.7554/eLife.07295.005

    Techniques Used: Recombinant, Labeling, Negative Control, Derivative Assay, Binding Assay, Expressing, Plasmid Preparation

    Conserved DNA elements within the 500 bp WRKY33 binding peak summit regions identified by MEME. ( A ) W-box with 5′ extended motifs. ( B ) W-box with 3′ extended motifs. ( C ) Additional conserved sequence GACTT/ATTC element. ( D ) Venn diagram illustrating the number of overlapping peaks containing both the W-box and the newly identified motif T/GTTGAAT. DOI: http://dx.doi.org/10.7554/eLife.07295.004
    Figure Legend Snippet: Conserved DNA elements within the 500 bp WRKY33 binding peak summit regions identified by MEME. ( A ) W-box with 5′ extended motifs. ( B ) W-box with 3′ extended motifs. ( C ) Additional conserved sequence GACTT/ATTC element. ( D ) Venn diagram illustrating the number of overlapping peaks containing both the W-box and the newly identified motif T/GTTGAAT. DOI: http://dx.doi.org/10.7554/eLife.07295.004

    Techniques Used: Binding Assay, Sequencing

    10) Product Images from "DNA-binding sequence specificity of DUX4"

    Article Title: DNA-binding sequence specificity of DUX4

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-016-0080-z

    Evaluating protein-DNA interaction by electrophoretic mobility shift. a Direct band shift assays for various sequence motifs. The DNA is detected by EtBr staining. The CT probe contains the control TAAT CT AATCA sequence and shows a complete shift to the slower-migrating form in the presence of an excess quantity of the DUX4 DNA-binding domain. Other oligos showed various shift efficiencies. b Competitive band shifts of the four flavors of the DUX4 ChIP-seq consensus motif. The CT probe is labelled with FAM; thus, the gel has no EtBr, and binding is competed with unlabeled oligos. All four flavors of the DUX4 ChIP-seq consensus effectively compete away binding to the FAM-CT probe when provided in excess, while the mutant sequence fails to compete. c Competitive band shifts of the sequences tested for direct band shifting (in a , above)
    Figure Legend Snippet: Evaluating protein-DNA interaction by electrophoretic mobility shift. a Direct band shift assays for various sequence motifs. The DNA is detected by EtBr staining. The CT probe contains the control TAAT CT AATCA sequence and shows a complete shift to the slower-migrating form in the presence of an excess quantity of the DUX4 DNA-binding domain. Other oligos showed various shift efficiencies. b Competitive band shifts of the four flavors of the DUX4 ChIP-seq consensus motif. The CT probe is labelled with FAM; thus, the gel has no EtBr, and binding is competed with unlabeled oligos. All four flavors of the DUX4 ChIP-seq consensus effectively compete away binding to the FAM-CT probe when provided in excess, while the mutant sequence fails to compete. c Competitive band shifts of the sequences tested for direct band shifting (in a , above)

    Techniques Used: Electrophoretic Mobility Shift Assay, Sequencing, Staining, Binding Assay, Chromatin Immunoprecipitation, Mutagenesis

    Evaluating reporters with multiple DUX4 binding sequences. a Dose-dependent activity of reporters with 1–24 copies of the CT motif at 100 ng of DNA/well. Note the logarithmic scale. b Dose-dependent activity of the same reporters at 4 ng/well. Synergy is demonstrated by the presence of activation with the 2× reporter but its absence with the 1× reporter at this concentration, as well as the continued increase in activity as copy number increases to 24×. c Transcriptional activity of reporters with two CT motifs spaced at different distances (indicated), compared to a reporter with a single CT motif
    Figure Legend Snippet: Evaluating reporters with multiple DUX4 binding sequences. a Dose-dependent activity of reporters with 1–24 copies of the CT motif at 100 ng of DNA/well. Note the logarithmic scale. b Dose-dependent activity of the same reporters at 4 ng/well. Synergy is demonstrated by the presence of activation with the 2× reporter but its absence with the 1× reporter at this concentration, as well as the continued increase in activity as copy number increases to 24×. c Transcriptional activity of reporters with two CT motifs spaced at different distances (indicated), compared to a reporter with a single CT motif

    Techniques Used: Binding Assay, Activity Assay, Activation Assay, Concentration Assay

    Discovery of DUX4-interacting motifs by SELEX. a PCR assay after indicated rounds of DNA pulldown. Filled triangle indicates the specific band; open triangle indicates primer dimers. Note that the amplified product is enriched over primer dimers by the last cycle shown. b Selection bias for each base at each position (base frequency in the pulled down sequence/base frequency in the control). c Luciferase reporter assays for the top four SELEX-identified sequences. d Direct band shift assays for the top 4 SELEX motifs. e Competitive activity of the top four SELEX motifs. Each unlabeled SELEX probe was used to compete the shift of a FAM-labeled probe bearing the CT motif
    Figure Legend Snippet: Discovery of DUX4-interacting motifs by SELEX. a PCR assay after indicated rounds of DNA pulldown. Filled triangle indicates the specific band; open triangle indicates primer dimers. Note that the amplified product is enriched over primer dimers by the last cycle shown. b Selection bias for each base at each position (base frequency in the pulled down sequence/base frequency in the control). c Luciferase reporter assays for the top four SELEX-identified sequences. d Direct band shift assays for the top 4 SELEX motifs. e Competitive activity of the top four SELEX motifs. Each unlabeled SELEX probe was used to compete the shift of a FAM-labeled probe bearing the CT motif

    Techniques Used: Polymerase Chain Reaction, Amplification, Selection, Sequencing, Luciferase, Electrophoretic Mobility Shift Assay, Activity Assay, Labeling

    11) Product Images from "Identification of alternative splicing events by RNA sequencing in early growth tomato fruits"

    Article Title: Identification of alternative splicing events by RNA sequencing in early growth tomato fruits

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2128-6

    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5
    Figure Legend Snippet: Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Negative Control, Polymerase Chain Reaction, Marker

    12) Product Images from "A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis"

    Article Title: A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis

    Journal: Scientific Reports

    doi: 10.1038/srep25340

    ObTLP1 -expressing Arabidopsis plants are tolerant to S. sclerotiorum infection. ( A ) Leaves of four-week-old ObTLP1 -expressing transgenic lines (TH1 and TH2) and vector control plants were inoculated with equal amount of mycelial suspension of S. sclerotiorum as described in Methods. Disease assessment was carried out at different time intervals and photographs were taken. ( B ) Lesion size was measured at different time intervals in infected leaves. Data are mean ± s.d. from sixteen plants for each transgenic line. ( C ) Fungal biomass in infected leaves was quantified by qPCR. Relative level of S. sclerotiorum ITS genomic DNA was determined using Arabidopsis chloroplast-encoded ribulose-1,5-bis-phosphate carboxylase/oxygenase large subunit as reference gene. Data are mean ± s.d. from three biological replicates. Asterisks indicate statistically significant difference at **P
    Figure Legend Snippet: ObTLP1 -expressing Arabidopsis plants are tolerant to S. sclerotiorum infection. ( A ) Leaves of four-week-old ObTLP1 -expressing transgenic lines (TH1 and TH2) and vector control plants were inoculated with equal amount of mycelial suspension of S. sclerotiorum as described in Methods. Disease assessment was carried out at different time intervals and photographs were taken. ( B ) Lesion size was measured at different time intervals in infected leaves. Data are mean ± s.d. from sixteen plants for each transgenic line. ( C ) Fungal biomass in infected leaves was quantified by qPCR. Relative level of S. sclerotiorum ITS genomic DNA was determined using Arabidopsis chloroplast-encoded ribulose-1,5-bis-phosphate carboxylase/oxygenase large subunit as reference gene. Data are mean ± s.d. from three biological replicates. Asterisks indicate statistically significant difference at **P

    Techniques Used: Expressing, Infection, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    ObTLP1 -expressing Arabidopsis plants are tolerant to B. cinerea infection. ( A ) Leaves of four-week-old ObTLP1 -expressing transgenic lines (TH1 and TH2) and vector control plants were inoculated with equal amount of spore suspension of B. cinerea as described in Methods. Disease assessment was carried out at different time intervals and photographs were taken. ( B ) Lesion size was measured at different time intervals in infected leaves. Data are mean ± s.d. from sixteen (vector control) or eight ( ObTLP1 -expressing lines) plants. ( C ) Fungal biomass in infected leaves was quantified by qPCR. Relative level of B. cinerea Actin genomic DNA was determined using Arabidopsis chloroplast-encoded ribulose-1,5-bis-phosphate carboxylase/oxygenase large subunit as reference gene. Data are mean ± s.d. from three biological replicates. Asterisks indicate statistically significant difference at **P
    Figure Legend Snippet: ObTLP1 -expressing Arabidopsis plants are tolerant to B. cinerea infection. ( A ) Leaves of four-week-old ObTLP1 -expressing transgenic lines (TH1 and TH2) and vector control plants were inoculated with equal amount of spore suspension of B. cinerea as described in Methods. Disease assessment was carried out at different time intervals and photographs were taken. ( B ) Lesion size was measured at different time intervals in infected leaves. Data are mean ± s.d. from sixteen (vector control) or eight ( ObTLP1 -expressing lines) plants. ( C ) Fungal biomass in infected leaves was quantified by qPCR. Relative level of B. cinerea Actin genomic DNA was determined using Arabidopsis chloroplast-encoded ribulose-1,5-bis-phosphate carboxylase/oxygenase large subunit as reference gene. Data are mean ± s.d. from three biological replicates. Asterisks indicate statistically significant difference at **P

    Techniques Used: Expressing, Infection, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    13) Product Images from "LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity"

    Article Title: LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity

    Journal: Scientific Reports

    doi: 10.1038/srep36741

    Effects of deletions within the ENII/BCP sequence on down-regulation of the ENII/BCP activity mediated by LUC7L3. ( A ) Deletion constructs of the ENII/BCP region of the HBV genome linked to the firefly luciferase gene are shown. Cells were co-transfected with one of these reporter constructs and the LUC7L3-expressing plasmid or an empty vector. At 48 h post-transfection, cells were harvested and subjected to the luciferase assay. Values obtained without LUC7L3 expression were set as 1 for expression of each reporter. Results represent the means with SEM from three independent transfectants. ( B ) Interaction of LUC7L3 with the ENII/BCP sequence but not with its deletion, del4 as indicated in ( A ), was shown by DNA pull-down assay. In vitro synthesized biotinylated DNA derived from entire ENII/BCP or its deletion of nt 1666–1700 region was mixed with the nuclear extract prepared from cells transfected with LUC7L3 construct. Biotinylated DNA-protein complexes were captured with streptavidin-conjugated beads, followed by immunoblotting with anti-FLAG antibody. ( C ) Effects of knockdown of HNF4α or C/EBPα on down-regulation of the ENII/BCP activity and the pgRNA expression were evaluated. At 24 h after introducing siRNA against HNF4α (siHNF4a) or C/EBPα (siC/EBPa) or negative control (siCont), cells were transfected with pUC-HB-Ce or pGLHBp1627/1817 together with the LUC7L3-expressing plasmid (LUC7) or an empty vector (EV) and cultured for further 48 h. The reporter activities (upper left) and the pgRNA levels (upper right) were determined by the luciferase assay and RT-qPCR, respectively. Knockdown efficiency of HNF4α (lower left) and C/EBPα (lower right) was assessed by RT-qPCR. Results represent the means with SEM from three independent transfectants.
    Figure Legend Snippet: Effects of deletions within the ENII/BCP sequence on down-regulation of the ENII/BCP activity mediated by LUC7L3. ( A ) Deletion constructs of the ENII/BCP region of the HBV genome linked to the firefly luciferase gene are shown. Cells were co-transfected with one of these reporter constructs and the LUC7L3-expressing plasmid or an empty vector. At 48 h post-transfection, cells were harvested and subjected to the luciferase assay. Values obtained without LUC7L3 expression were set as 1 for expression of each reporter. Results represent the means with SEM from three independent transfectants. ( B ) Interaction of LUC7L3 with the ENII/BCP sequence but not with its deletion, del4 as indicated in ( A ), was shown by DNA pull-down assay. In vitro synthesized biotinylated DNA derived from entire ENII/BCP or its deletion of nt 1666–1700 region was mixed with the nuclear extract prepared from cells transfected with LUC7L3 construct. Biotinylated DNA-protein complexes were captured with streptavidin-conjugated beads, followed by immunoblotting with anti-FLAG antibody. ( C ) Effects of knockdown of HNF4α or C/EBPα on down-regulation of the ENII/BCP activity and the pgRNA expression were evaluated. At 24 h after introducing siRNA against HNF4α (siHNF4a) or C/EBPα (siC/EBPa) or negative control (siCont), cells were transfected with pUC-HB-Ce or pGLHBp1627/1817 together with the LUC7L3-expressing plasmid (LUC7) or an empty vector (EV) and cultured for further 48 h. The reporter activities (upper left) and the pgRNA levels (upper right) were determined by the luciferase assay and RT-qPCR, respectively. Knockdown efficiency of HNF4α (lower left) and C/EBPα (lower right) was assessed by RT-qPCR. Results represent the means with SEM from three independent transfectants.

    Techniques Used: Sequencing, Activity Assay, Construct, Luciferase, Transfection, Expressing, Plasmid Preparation, Pull Down Assay, In Vitro, Synthesized, Derivative Assay, Negative Control, Cell Culture, Quantitative RT-PCR

    14) Product Images from "Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China"

    Article Title: Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China

    Journal: Scientific Reports

    doi: 10.1038/srep31075

    Electropherogram of Control DNA 007 (1 ng) amplified by Huaxia Platinum System. Control DNA 007 was amplified following the recommended protocol (27 cycles). Amplified product was separated on an Applied Biosystems 3500 Genetic Analyzer. Panel labeled “Yin…” is Y-InDel. Panel labeled “AM…” is Amelogenin.
    Figure Legend Snippet: Electropherogram of Control DNA 007 (1 ng) amplified by Huaxia Platinum System. Control DNA 007 was amplified following the recommended protocol (27 cycles). Amplified product was separated on an Applied Biosystems 3500 Genetic Analyzer. Panel labeled “Yin…” is Y-InDel. Panel labeled “AM…” is Amelogenin.

    Techniques Used: Amplification, Labeling

    15) Product Images from "Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China"

    Article Title: Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China

    Journal: Scientific Reports

    doi: 10.1038/srep31075

    Electropherogram of Control DNA 007 (1 ng) amplified by Huaxia Platinum System. Control DNA 007 was amplified following the recommended protocol (27 cycles). Amplified product was separated on an Applied Biosystems 3500 Genetic Analyzer. Panel labeled “Yin…” is Y-InDel. Panel labeled “AM…” is Amelogenin.
    Figure Legend Snippet: Electropherogram of Control DNA 007 (1 ng) amplified by Huaxia Platinum System. Control DNA 007 was amplified following the recommended protocol (27 cycles). Amplified product was separated on an Applied Biosystems 3500 Genetic Analyzer. Panel labeled “Yin…” is Y-InDel. Panel labeled “AM…” is Amelogenin.

    Techniques Used: Amplification, Labeling

    16) Product Images from "Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine"

    Article Title: Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw473

    Effects of 5′ and 3′ phosphorothioate bonds on the magnitude of inhibition of the EGFP gene expression by single synthetic 8-oxodG in HeLa cells. ( A ) Representative EGFP fluorescence distribution plots of cells transfected with vectors containing 8-oxodG in combination with the specified DNA backbone modifications (amber lines). Cells in the reference sample (overlaid blue line) were transfected with a control vector containing the corresponding synthetic oligonucleotide without 8-oxoG. ( B ) EGFP expression in cells transfected with the specified constructs, each normalised relative to the reference construct without 8-oxoG, transfected in parallel. Summary of 6 independent experiments (mean ± SD). P -values: Student's two-tailed t -test. ( C ) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) analysed at 24 h post-transfection, as described in (A). Error bars show data range ( n = 2). ( D ) DNA strand cleavage activities in the OGG1-overexpressing cell extracts towards plasmid substrates containing the specified modifications. Agarose gel and quantification of the nicked fraction.
    Figure Legend Snippet: Effects of 5′ and 3′ phosphorothioate bonds on the magnitude of inhibition of the EGFP gene expression by single synthetic 8-oxodG in HeLa cells. ( A ) Representative EGFP fluorescence distribution plots of cells transfected with vectors containing 8-oxodG in combination with the specified DNA backbone modifications (amber lines). Cells in the reference sample (overlaid blue line) were transfected with a control vector containing the corresponding synthetic oligonucleotide without 8-oxoG. ( B ) EGFP expression in cells transfected with the specified constructs, each normalised relative to the reference construct without 8-oxoG, transfected in parallel. Summary of 6 independent experiments (mean ± SD). P -values: Student's two-tailed t -test. ( C ) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) analysed at 24 h post-transfection, as described in (A). Error bars show data range ( n = 2). ( D ) DNA strand cleavage activities in the OGG1-overexpressing cell extracts towards plasmid substrates containing the specified modifications. Agarose gel and quantification of the nicked fraction.

    Techniques Used: Inhibition, Expressing, Fluorescence, Transfection, Plasmid Preparation, Construct, Two Tailed Test, Agarose Gel Electrophoresis

    17) Product Images from "Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells"

    Article Title: Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023667

    SiPS generation from SMs by single small molecule treatment. (A) The native SMs cells were stained for desmin expression (red fluorescence) and confirmed by flow cytometry (96%) to show the purity of SMs. The nuclei were stained with DAPI (blue fluorescence). (B) Gene expression profile of various stemness and myogenic markers in SMs, ES cells and SiPS isolates. RT-PCR analyses showing the endogenous expression of myogenic and pluripotency marker genes in SMs and ES cells. SMs showed 3 pluripotency markers (Sox2, Klf4, cMyc) in addition to MyoD and Pax7 in comparison to ES cell expressing all 4 pluripotency markers. RG108 significantly induced Oct3/4 expression in SiPS with simultaneous loss of myogenic markers MyoD and Pax7 after reprogramming. Densitometric quantization of mRNA expression of Oct3/4, Sox2, Klf4, cMyc, MyoD and Pax7 in SMs, ES cells and SiPS are given in Figure S1 . (C) Chemical structure of RG108 (D) Phase contrast and (E) fluorescent images of GFP expressing SiPS clones generated by RG108 (500 µM). (F) Graph showing generation of approximately 57–60 GFP + clones from 50,000 SMs 2–3 weeks after RG108 treatment. The control SMs were either without treatment or treated with DMSO (the solvent for RG108). Similar results were obtained in three independent experiments. (G) DNA methylation analysis showing significant inhibition of DNA methyltransferase activity in SiPS cells in comparison to the untreated SMs. Positive control shown in the graph was provided with the assay kit.
    Figure Legend Snippet: SiPS generation from SMs by single small molecule treatment. (A) The native SMs cells were stained for desmin expression (red fluorescence) and confirmed by flow cytometry (96%) to show the purity of SMs. The nuclei were stained with DAPI (blue fluorescence). (B) Gene expression profile of various stemness and myogenic markers in SMs, ES cells and SiPS isolates. RT-PCR analyses showing the endogenous expression of myogenic and pluripotency marker genes in SMs and ES cells. SMs showed 3 pluripotency markers (Sox2, Klf4, cMyc) in addition to MyoD and Pax7 in comparison to ES cell expressing all 4 pluripotency markers. RG108 significantly induced Oct3/4 expression in SiPS with simultaneous loss of myogenic markers MyoD and Pax7 after reprogramming. Densitometric quantization of mRNA expression of Oct3/4, Sox2, Klf4, cMyc, MyoD and Pax7 in SMs, ES cells and SiPS are given in Figure S1 . (C) Chemical structure of RG108 (D) Phase contrast and (E) fluorescent images of GFP expressing SiPS clones generated by RG108 (500 µM). (F) Graph showing generation of approximately 57–60 GFP + clones from 50,000 SMs 2–3 weeks after RG108 treatment. The control SMs were either without treatment or treated with DMSO (the solvent for RG108). Similar results were obtained in three independent experiments. (G) DNA methylation analysis showing significant inhibition of DNA methyltransferase activity in SiPS cells in comparison to the untreated SMs. Positive control shown in the graph was provided with the assay kit.

    Techniques Used: Staining, Expressing, Fluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Marker, Clone Assay, Generated, DNA Methylation Assay, Inhibition, Activity Assay, Positive Control

    18) Product Images from "XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima"

    Article Title: XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150292

    Identification of the X chromosome in the Strigamia karyotype by FISH with a set of X-chromosome derived DNA probes. (A) The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. (B) A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the Strigamia karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). (C) Two Strigamia karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in Fig 1A . We infer that the pair of sex chromosomes represents the 4 th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. (D) Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. (E) A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). (F) Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).
    Figure Legend Snippet: Identification of the X chromosome in the Strigamia karyotype by FISH with a set of X-chromosome derived DNA probes. (A) The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. (B) A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the Strigamia karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). (C) Two Strigamia karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in Fig 1A . We infer that the pair of sex chromosomes represents the 4 th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. (D) Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. (E) A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). (F) Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).

    Techniques Used: Fluorescence In Situ Hybridization, Derivative Assay, Polymerase Chain Reaction, Hybridization, Construct, Staining

    19) Product Images from "Frequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan"

    Article Title: Frequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155520

    Quantitative real-time PCR analysis of coinfected cotton plants. Graphs show the determined titers of (A) virus ( Tomato leaf curl New Delhi virus [ToLCNDV] DNA-A and Cotton leaf curl Kokhran virus -Burewala CLCuKoV-Bur), (B) ToLCNDV DNA-B and (C) Cotton leaf curl Multan betasatellite (CLCuMB). The DNA samples used in the PCR reactions were extracted from a healthy cotton plant (H.C), tomato plants infected with ToLCNDV (T1 and T2), field collected cotton plants with severe cotton leaf curl disease symptoms either infected with only CLCuKoV-Bur/CLCuMB (C-1 and C-2) or coinfected with CLCuKoV-Bur/CLCuMB and ToLCNDV (C-4 to C-7). The titer of each component is given in ng/μg of genomic DNA on the y-axis and is the mean of three replications. The error bars are the divergence from mean quantified value.
    Figure Legend Snippet: Quantitative real-time PCR analysis of coinfected cotton plants. Graphs show the determined titers of (A) virus ( Tomato leaf curl New Delhi virus [ToLCNDV] DNA-A and Cotton leaf curl Kokhran virus -Burewala CLCuKoV-Bur), (B) ToLCNDV DNA-B and (C) Cotton leaf curl Multan betasatellite (CLCuMB). The DNA samples used in the PCR reactions were extracted from a healthy cotton plant (H.C), tomato plants infected with ToLCNDV (T1 and T2), field collected cotton plants with severe cotton leaf curl disease symptoms either infected with only CLCuKoV-Bur/CLCuMB (C-1 and C-2) or coinfected with CLCuKoV-Bur/CLCuMB and ToLCNDV (C-4 to C-7). The titer of each component is given in ng/μg of genomic DNA on the y-axis and is the mean of three replications. The error bars are the divergence from mean quantified value.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Infection

    Phylogenetic analysis of the sequences of begomoviruses, and satellites obtained in the study. Trees were constructed from alignments of the sequences of (A and F) Cotton leaf curl Kokhran virus (CLCuKoV), (B and G) Cotton leaf curl Multan betasatellite (CLCuMB), (C) Cotton leaf curl Multan alphasatellite (CLCuMA), (D) Tomato leaf curl New Delhi virus (ToLCNDV) DNA-B and (E) ToLCNDV DNA-A using the Neighbor-Joining method. Isolates in green were obtained in the study described here. The numbers at nodes represent percentage bootstrap scores (1000 replicates). The strain descriptors (in square brackets) in each case give country, location, host and year of sampling. For each isolate the database accession number is given. The trees were arbitrarily rooted on the sequence of Tomato mottle virus (ToMoV) (A, D and E), CLCuMA (B) and CLCuMB (C) as outgroup. The strains of CLCuKoV given in panel F are Burewala (Bur), Layyah (Lay), Lucknow (Luc), Kokhran (Kok) and Shadadpur (Sha). The strains of CLCuMB given in panel G are given as Burewala (Bur), Multan (Mul) and Shadadpur (Sha).
    Figure Legend Snippet: Phylogenetic analysis of the sequences of begomoviruses, and satellites obtained in the study. Trees were constructed from alignments of the sequences of (A and F) Cotton leaf curl Kokhran virus (CLCuKoV), (B and G) Cotton leaf curl Multan betasatellite (CLCuMB), (C) Cotton leaf curl Multan alphasatellite (CLCuMA), (D) Tomato leaf curl New Delhi virus (ToLCNDV) DNA-B and (E) ToLCNDV DNA-A using the Neighbor-Joining method. Isolates in green were obtained in the study described here. The numbers at nodes represent percentage bootstrap scores (1000 replicates). The strain descriptors (in square brackets) in each case give country, location, host and year of sampling. For each isolate the database accession number is given. The trees were arbitrarily rooted on the sequence of Tomato mottle virus (ToMoV) (A, D and E), CLCuMA (B) and CLCuMB (C) as outgroup. The strains of CLCuKoV given in panel F are Burewala (Bur), Layyah (Lay), Lucknow (Luc), Kokhran (Kok) and Shadadpur (Sha). The strains of CLCuMB given in panel G are given as Burewala (Bur), Multan (Mul) and Shadadpur (Sha).

    Techniques Used: Construct, Sampling, Sequencing

    20) Product Images from "Evaluation of a Salmonella Strain Lacking the Secondary Messenger C-di-GMP and RpoS as a Live Oral Vaccine"

    Article Title: Evaluation of a Salmonella Strain Lacking the Secondary Messenger C-di-GMP and RpoS as a Live Oral Vaccine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161216

    ΔXIII is a DIVA vaccine that allows differentiation of infected and vaccinated animals. (A) SEN4316 based ELISA of pooled sera from wild type and ΔXIII infected animals, obtained before infection (PreIM) and 33 and 44 days post infection. Sera from animals infected with the wild type strain and not with ΔXIII present antibodies against the SEN4316 protein. Error bars represent standard deviation between triplicate wells. (B) DNA from pooled faecal samples from wild type and ΔXIII infected animals, collected at day 1, 7, 14 and 21 post infection, were analyzed by PCR. Amplification of stool DNA with primers DIVA-1 and DIVA-2 allowed the identification of vaccinated animals with ΔXIII strain. Amplification with primers invA-fw and invA-rv served as a control of the presence of Salmonella DNA. Results shown are representative of results obtained throughout time, since Salmonella DNA was present in faecal samples since day one post infection.
    Figure Legend Snippet: ΔXIII is a DIVA vaccine that allows differentiation of infected and vaccinated animals. (A) SEN4316 based ELISA of pooled sera from wild type and ΔXIII infected animals, obtained before infection (PreIM) and 33 and 44 days post infection. Sera from animals infected with the wild type strain and not with ΔXIII present antibodies against the SEN4316 protein. Error bars represent standard deviation between triplicate wells. (B) DNA from pooled faecal samples from wild type and ΔXIII infected animals, collected at day 1, 7, 14 and 21 post infection, were analyzed by PCR. Amplification of stool DNA with primers DIVA-1 and DIVA-2 allowed the identification of vaccinated animals with ΔXIII strain. Amplification with primers invA-fw and invA-rv served as a control of the presence of Salmonella DNA. Results shown are representative of results obtained throughout time, since Salmonella DNA was present in faecal samples since day one post infection.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Polymerase Chain Reaction, Amplification

    21) Product Images from "Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis"

    Article Title: Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05945-4

    Hox gene expression correlates with Hox cluster chromatin opening. a Schematic representation of ANT-C and BX-C (Hox genes in blue, positions of DNA FISH probes in red, and positions of RNA FISH probes in green; the arrows show promoters). b Schematic diagram depicting the PSs of Drosophila embryos in which measurements were performed. c Relative density of RNA FISH spots measured for each Hox gene along the anteroposterior axis of WT Drosophila embryos 3:50–7:20 after fertilization. The density of RNA FISH spots is calculated by dividing the number of spots by the area of the PS. d , e Distances between the promoters of Ubx , abdA , and AbdB (d) or lab , Scr , and Antp (e) measured within cell nuclei of WT Drosophila embryos 3:50–7:20 after fertilization along the anteroposterior axis. We calculated distances between centroids of DNA FISH spots in three dimensions. For each PS of one embryo, we computed the median distances Ubx – abdA , abdA – AbdB , Ubx – AbdB or lab – Scr , Scr – Antp , lab – Antp . Curves represent the average median distances measured in several embryos and the corresponding error bar (SEM; N ≥ 16). f , g Heat maps showing correlations between Hox gene transcription and chromatin opening in the BX-C ( f ) and the ANT-C ( g ). The physical distance (Dist) between two loci within a Hox complex measured the chromatin opening and Hox gene expression (Exp) is calculated by adding the relative density of RNA FISH spot of Hox genes comprised between these two loci. All measurements were normalized between their minimum and their maximum. r indicates correlation coefficients. h , i Distances between promoters of Ubx , abdA , and AbdB (h) or lab , Scr and Antp (i) and the closest Polycomb foci were measured in WT Drosophila embryos 3:50–7:20 after fertilization. The percentage of distances measuring less than 400 nm was calculated for each PS of one embryo. Curves represent the mean percentage measured in several embryos. The highlighted PSs show regions wherein Hox genes were found to be significantly further away from Pc foci than in Head-PS0 ( t -test, one-tailed, P
    Figure Legend Snippet: Hox gene expression correlates with Hox cluster chromatin opening. a Schematic representation of ANT-C and BX-C (Hox genes in blue, positions of DNA FISH probes in red, and positions of RNA FISH probes in green; the arrows show promoters). b Schematic diagram depicting the PSs of Drosophila embryos in which measurements were performed. c Relative density of RNA FISH spots measured for each Hox gene along the anteroposterior axis of WT Drosophila embryos 3:50–7:20 after fertilization. The density of RNA FISH spots is calculated by dividing the number of spots by the area of the PS. d , e Distances between the promoters of Ubx , abdA , and AbdB (d) or lab , Scr , and Antp (e) measured within cell nuclei of WT Drosophila embryos 3:50–7:20 after fertilization along the anteroposterior axis. We calculated distances between centroids of DNA FISH spots in three dimensions. For each PS of one embryo, we computed the median distances Ubx – abdA , abdA – AbdB , Ubx – AbdB or lab – Scr , Scr – Antp , lab – Antp . Curves represent the average median distances measured in several embryos and the corresponding error bar (SEM; N ≥ 16). f , g Heat maps showing correlations between Hox gene transcription and chromatin opening in the BX-C ( f ) and the ANT-C ( g ). The physical distance (Dist) between two loci within a Hox complex measured the chromatin opening and Hox gene expression (Exp) is calculated by adding the relative density of RNA FISH spot of Hox genes comprised between these two loci. All measurements were normalized between their minimum and their maximum. r indicates correlation coefficients. h , i Distances between promoters of Ubx , abdA , and AbdB (h) or lab , Scr and Antp (i) and the closest Polycomb foci were measured in WT Drosophila embryos 3:50–7:20 after fertilization. The percentage of distances measuring less than 400 nm was calculated for each PS of one embryo. Curves represent the mean percentage measured in several embryos. The highlighted PSs show regions wherein Hox genes were found to be significantly further away from Pc foci than in Head-PS0 ( t -test, one-tailed, P

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, One-tailed Test

    22) Product Images from "Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest"

    Article Title: Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.211

    DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. ( a ) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α -Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. ( b ) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μ M) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β -Tubulin or GAPDH was used as loading controls. ( c ) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μ M continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. ( d ) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μ M of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μ M). A representative blot of two independent experiments is shown. GAPDH was used as a loading control
    Figure Legend Snippet: DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. ( a ) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α -Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. ( b ) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μ M) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β -Tubulin or GAPDH was used as loading controls. ( c ) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μ M continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. ( d ) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μ M of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μ M). A representative blot of two independent experiments is shown. GAPDH was used as a loading control

    Techniques Used: Activation Assay, Western Blot

    23) Product Images from "Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques"

    Article Title: Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202997

    Vaccines targeting SIV protease cleavage sites (PCSs) or full Gag and Env proteins. (A) Diagram of the twelve protease cleavage sites (PCS1 through PCS12), located on three SIV polyproteins (Pr55Gag, Pr160Gag-Pol and Nef precursor), not drawn to scale. MA: matrix; CA: capsid; NC: nucleocapsid; TFP: transframe protein; PR: protease; RT: reverse transcriptase; and IN: integrase. (B) Peptide sequences of SIV immunogens in a conserved element vaccine targeting the PCSs (the PCS vaccine). Each sequence corresponds to -10 through +10 amino acid positions flanking each cleavage site. Slash (/) indicates the site of protease cleavage. These sequences were confirmed to be specific for SIV by NCBI protein BLAST and conserved among multiple SIV strains. The peptide immunogens were delivered as recombinant vesicular stomatitis viruses (rVSVpcs) and nanoparticles (NANOpcs). Peptide antigens with these sequences were also used in a Bio-Plex multiplexed assay to detect anti-PCS antibodies. (C) Sequences of three Gag or Env (non-PCS) peptides used in Bio-Plex to detect anti-Gag or Env antibodies, including one Gag peptide, named SIVgag, and two Env peptides, named SIVenv1 and SIVenv2. (D) Western blot analyses of protein expression from a full-length Gag and Env-based vaccine (the Gag/Env vaccine). VeroE6 cells were infected with recombinant vesicular stomatitis viruses (rVSVs) carrying full Gag or Env gene of SIVmac239 (rVSVgag/env) and the culture supernatants were analyzed by Western blot to detect Gag or Env protein expression using standard monoclonal antibodies (mAb, NIH AIDS Reagent Program) to Gag or Env. The full Gag and Env genes were also cloned into pVAX1 (a DNA vaccine vector), respectively, followed by NANO packaging (NANOgag/env). HEK293T cells were transfected with these DNA vaccines and analyzed by Western blot. (E) Vaccination scheme. Three groups (Control, PCS vaccine and Gag/Env vaccine) of eight female MCMs per group were primed and boosted on indicated weeks (wk). The Control group received empty rVSV virus and sterile water. One animal from the Gag/Env vaccine group was euthanized early due to severe health issues unrelated to vaccination, leaving seven animals in this group to complete the study. rVSV control vector (rVSV), rVSVpcs or rVSVgag/env was administered intramuscularly. NANO control vector (sterile water), NANOpcs or NANOgag/env was administered intranasally.
    Figure Legend Snippet: Vaccines targeting SIV protease cleavage sites (PCSs) or full Gag and Env proteins. (A) Diagram of the twelve protease cleavage sites (PCS1 through PCS12), located on three SIV polyproteins (Pr55Gag, Pr160Gag-Pol and Nef precursor), not drawn to scale. MA: matrix; CA: capsid; NC: nucleocapsid; TFP: transframe protein; PR: protease; RT: reverse transcriptase; and IN: integrase. (B) Peptide sequences of SIV immunogens in a conserved element vaccine targeting the PCSs (the PCS vaccine). Each sequence corresponds to -10 through +10 amino acid positions flanking each cleavage site. Slash (/) indicates the site of protease cleavage. These sequences were confirmed to be specific for SIV by NCBI protein BLAST and conserved among multiple SIV strains. The peptide immunogens were delivered as recombinant vesicular stomatitis viruses (rVSVpcs) and nanoparticles (NANOpcs). Peptide antigens with these sequences were also used in a Bio-Plex multiplexed assay to detect anti-PCS antibodies. (C) Sequences of three Gag or Env (non-PCS) peptides used in Bio-Plex to detect anti-Gag or Env antibodies, including one Gag peptide, named SIVgag, and two Env peptides, named SIVenv1 and SIVenv2. (D) Western blot analyses of protein expression from a full-length Gag and Env-based vaccine (the Gag/Env vaccine). VeroE6 cells were infected with recombinant vesicular stomatitis viruses (rVSVs) carrying full Gag or Env gene of SIVmac239 (rVSVgag/env) and the culture supernatants were analyzed by Western blot to detect Gag or Env protein expression using standard monoclonal antibodies (mAb, NIH AIDS Reagent Program) to Gag or Env. The full Gag and Env genes were also cloned into pVAX1 (a DNA vaccine vector), respectively, followed by NANO packaging (NANOgag/env). HEK293T cells were transfected with these DNA vaccines and analyzed by Western blot. (E) Vaccination scheme. Three groups (Control, PCS vaccine and Gag/Env vaccine) of eight female MCMs per group were primed and boosted on indicated weeks (wk). The Control group received empty rVSV virus and sterile water. One animal from the Gag/Env vaccine group was euthanized early due to severe health issues unrelated to vaccination, leaving seven animals in this group to complete the study. rVSV control vector (rVSV), rVSVpcs or rVSVgag/env was administered intramuscularly. NANO control vector (sterile water), NANOpcs or NANOgag/env was administered intranasally.

    Techniques Used: Sequencing, Recombinant, Western Blot, Expressing, Infection, Clone Assay, Plasmid Preparation, Transfection

    24) Product Images from "IL-21 modulates memory and exhaustion phenotype of T-cells in a fatty acid oxidation-dependent manner"

    Article Title: IL-21 modulates memory and exhaustion phenotype of T-cells in a fatty acid oxidation-dependent manner

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24442

    IL-21 increases mitochondrial biogenesis and fitness ( A – F ) T-cell cultivation was performed over 5 days with IL-2 or IL-21 in the presence of activation/expansion beads. (A) Mitochondrial mass was visualized using MitoTracker™ in cytokine-treated T-cells by fluorescence microscopy as shown for one representative donor. Samples were also counterstained with the nuclear dye DAPI. Quantification of MitoTracker™ fluorescence was done with ImageJ for 5 single cells per condition. (B) The content of mtDNA was determined as the mitochondrial DNA copy number relative to nuclear DNA using qPCR analysis ( n = 6). (C) The mitochondrial superoxide production was semiquantified using MitoSOX™ probe with flow cytometry ( n = 6). (D) The mitochondrial membrane potential (∆ΨM) was semiquantified by flow cytometry using the potentiometric dye JC-1 ( n = 6). In mitochondria with a high membrane potential JC-1 forms aggregates which exhibit a fluorescence emission of higher wavelength (approx. 590 nm). In mitochondria with a low membrane potential JC-1 aggregates separate which feature a shift of the emission to lower wavelength (approx. 530 nm). Hereby, the ratio of both red and green fluorescence allows comparing the membrane potential between the two cell culture conditions. (E) Total intracellular ROS content was detected in IL-21 treated T-cells compared to IL-2 under both non-activating and activating conditions by flow cytometry ( n = 6). (F) The relative gene expression of key cellular antioxidants (catalase, CAT; heme-oxygenase-1, HMOX1; glutamate cysteine ligase catalytic subunit, GCLC; glutamate cysteine ligase modifier subunit, GCLM) is shown for IL-21 treated T-cells in relation to IL-2 treated counterparts as quantified by qPCR ( n = 6). ( G ) CLL PBMCs with less than 70% CD5 + B-cells were cultured for 5 days in the presence of IL-2 or IL-21 with anti-CD2/CD3/CD28 beads. Total cells were incubated with MitoSOX™ for mitochondrial superoxide production and CellROX™ for total intracellular ROS level. For analysis of T-cells, cells were gated according to their CD3 expression. MFI is defined as median fluorescence intensity. Error bars indicate the standard error means. P value: * P
    Figure Legend Snippet: IL-21 increases mitochondrial biogenesis and fitness ( A – F ) T-cell cultivation was performed over 5 days with IL-2 or IL-21 in the presence of activation/expansion beads. (A) Mitochondrial mass was visualized using MitoTracker™ in cytokine-treated T-cells by fluorescence microscopy as shown for one representative donor. Samples were also counterstained with the nuclear dye DAPI. Quantification of MitoTracker™ fluorescence was done with ImageJ for 5 single cells per condition. (B) The content of mtDNA was determined as the mitochondrial DNA copy number relative to nuclear DNA using qPCR analysis ( n = 6). (C) The mitochondrial superoxide production was semiquantified using MitoSOX™ probe with flow cytometry ( n = 6). (D) The mitochondrial membrane potential (∆ΨM) was semiquantified by flow cytometry using the potentiometric dye JC-1 ( n = 6). In mitochondria with a high membrane potential JC-1 forms aggregates which exhibit a fluorescence emission of higher wavelength (approx. 590 nm). In mitochondria with a low membrane potential JC-1 aggregates separate which feature a shift of the emission to lower wavelength (approx. 530 nm). Hereby, the ratio of both red and green fluorescence allows comparing the membrane potential between the two cell culture conditions. (E) Total intracellular ROS content was detected in IL-21 treated T-cells compared to IL-2 under both non-activating and activating conditions by flow cytometry ( n = 6). (F) The relative gene expression of key cellular antioxidants (catalase, CAT; heme-oxygenase-1, HMOX1; glutamate cysteine ligase catalytic subunit, GCLC; glutamate cysteine ligase modifier subunit, GCLM) is shown for IL-21 treated T-cells in relation to IL-2 treated counterparts as quantified by qPCR ( n = 6). ( G ) CLL PBMCs with less than 70% CD5 + B-cells were cultured for 5 days in the presence of IL-2 or IL-21 with anti-CD2/CD3/CD28 beads. Total cells were incubated with MitoSOX™ for mitochondrial superoxide production and CellROX™ for total intracellular ROS level. For analysis of T-cells, cells were gated according to their CD3 expression. MFI is defined as median fluorescence intensity. Error bars indicate the standard error means. P value: * P

    Techniques Used: Activation Assay, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Cell Culture, Expressing, Incubation

    25) Product Images from "Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine"

    Article Title: Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw473

    Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.
    Figure Legend Snippet: Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Inhibition, Ligation

    Deletion mutants of the CMV-IE promoter are susceptible to the inhibition of the reporter gene expression by single 8-oxoG. ( A ) List of vectors containing the modified CMV_1111 promoter or its truncated versions together with the map of the introduced deletions. Batons show the canonical CRE sequences. ( B ) Fluorescence distribution plots and quantification of EGFP expression in HeLa cells 24 h post-transfection with the specified promoter constructs without artificially introduced base modifications. ( C ) Overlaid EGFP fluorescence distribution plots of cells transfected with vectors containing synthetic DNA strand with one 8-oxoG (amber) and those containing the respective unmodified oligonucleotide (blue). ( D ) Impact of single 8-oxoG on the gene expression as a function of promoter strength. Data of three independent experiments and the best-fit linear regression lines. EGFP expression was measured at 6 and 24 h post-transfection and calculated relative to expression of the matched construct without 8-oxoG.
    Figure Legend Snippet: Deletion mutants of the CMV-IE promoter are susceptible to the inhibition of the reporter gene expression by single 8-oxoG. ( A ) List of vectors containing the modified CMV_1111 promoter or its truncated versions together with the map of the introduced deletions. Batons show the canonical CRE sequences. ( B ) Fluorescence distribution plots and quantification of EGFP expression in HeLa cells 24 h post-transfection with the specified promoter constructs without artificially introduced base modifications. ( C ) Overlaid EGFP fluorescence distribution plots of cells transfected with vectors containing synthetic DNA strand with one 8-oxoG (amber) and those containing the respective unmodified oligonucleotide (blue). ( D ) Impact of single 8-oxoG on the gene expression as a function of promoter strength. Data of three independent experiments and the best-fit linear regression lines. EGFP expression was measured at 6 and 24 h post-transfection and calculated relative to expression of the matched construct without 8-oxoG.

    Techniques Used: Inhibition, Expressing, Modification, Fluorescence, Transfection, Construct

    Inhibitory effect of single 8-oxoG on the expression of the inducible EGFP gene. ( A ) Scheme of the tetracycline-regulated (tet-on) EGFP expression vector. Two TetR binding motifs (TetO 2 ×2) were introduced into the 5′-untranslated region without affecting the protein-coding sequence. Tandem Nt.Bpu10I nicking sites were retained and used for incorporation of synthetic oligonucleotides, as above. ( B ) Verification of the incorporation of 8-oxoG into the tet-on vector. DNA strand scission analysis of constructs produced with unmodified synthetic oligonucleotide (G) or the oligonucleotide containing single 8-oxoG (8oG). ( C ) Representative fluorescence distribution plots of T-REx™-Hela cells 24 h post-transfection with the expression constructs containing synthetic oligonucleotides with or without 8-oxoG. Cells were incubated in the absence (- tet) or in the presence of tetracycline (+ tet). ( D ) Relative EGFP expression calculated at the uninduced (- tet) and induced (+ tet) conditions ( n = 6, ± SD).
    Figure Legend Snippet: Inhibitory effect of single 8-oxoG on the expression of the inducible EGFP gene. ( A ) Scheme of the tetracycline-regulated (tet-on) EGFP expression vector. Two TetR binding motifs (TetO 2 ×2) were introduced into the 5′-untranslated region without affecting the protein-coding sequence. Tandem Nt.Bpu10I nicking sites were retained and used for incorporation of synthetic oligonucleotides, as above. ( B ) Verification of the incorporation of 8-oxoG into the tet-on vector. DNA strand scission analysis of constructs produced with unmodified synthetic oligonucleotide (G) or the oligonucleotide containing single 8-oxoG (8oG). ( C ) Representative fluorescence distribution plots of T-REx™-Hela cells 24 h post-transfection with the expression constructs containing synthetic oligonucleotides with or without 8-oxoG. Cells were incubated in the absence (- tet) or in the presence of tetracycline (+ tet). ( D ) Relative EGFP expression calculated at the uninduced (- tet) and induced (+ tet) conditions ( n = 6, ± SD).

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay, Sequencing, Construct, Produced, Fluorescence, Transfection, Incubation

    Effects of 5′ and 3′ phosphorothioate bonds on the magnitude of inhibition of the EGFP gene expression by single synthetic 8-oxodG in HeLa cells. ( A ) Representative EGFP fluorescence distribution plots of cells transfected with vectors containing 8-oxodG in combination with the specified DNA backbone modifications (amber lines). Cells in the reference sample (overlaid blue line) were transfected with a control vector containing the corresponding synthetic oligonucleotide without 8-oxoG. ( B ) EGFP expression in cells transfected with the specified constructs, each normalised relative to the reference construct without 8-oxoG, transfected in parallel. Summary of 6 independent experiments (mean ± SD). P -values: Student's two-tailed t -test. ( C ) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) analysed at 24 h post-transfection, as described in (A). Error bars show data range ( n = 2). ( D ) DNA strand cleavage activities in the OGG1-overexpressing cell extracts towards plasmid substrates containing the specified modifications. Agarose gel and quantification of the nicked fraction.
    Figure Legend Snippet: Effects of 5′ and 3′ phosphorothioate bonds on the magnitude of inhibition of the EGFP gene expression by single synthetic 8-oxodG in HeLa cells. ( A ) Representative EGFP fluorescence distribution plots of cells transfected with vectors containing 8-oxodG in combination with the specified DNA backbone modifications (amber lines). Cells in the reference sample (overlaid blue line) were transfected with a control vector containing the corresponding synthetic oligonucleotide without 8-oxoG. ( B ) EGFP expression in cells transfected with the specified constructs, each normalised relative to the reference construct without 8-oxoG, transfected in parallel. Summary of 6 independent experiments (mean ± SD). P -values: Student's two-tailed t -test. ( C ) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) analysed at 24 h post-transfection, as described in (A). Error bars show data range ( n = 2). ( D ) DNA strand cleavage activities in the OGG1-overexpressing cell extracts towards plasmid substrates containing the specified modifications. Agarose gel and quantification of the nicked fraction.

    Techniques Used: Inhibition, Expressing, Fluorescence, Transfection, Plasmid Preparation, Construct, Two Tailed Test, Agarose Gel Electrophoresis

    Inhibition of the EGFP gene expression by single apurinic site (AP). ( A ) Vectors used for site-specific insertion of synthetic oligonucleotides containing a single AP lesion (tetrahydrofuran) on the place of dG in the transcribed (TS) or the non-transcribed (NTS) strand of the EGFP gene. ( B ) Activity of APE1 toward plasmid substrates containing single synthetic AP lesion and the effects of the specified phosphorothioate linkages. ( C ) Fluorescence distribution plots of HeLa cells 24 h post-transfection with the expression constructs containing the indicated modifications in the specified DNA strand (TS or NTS), compared to the ‘G’ construct (overlaid blue line). Analogous plots for cells incubated in parallel with PARP inhibitors are shown in Supplementary Figure S5. ( D ) Mean relative EGFP expression of the specified constructs in the absence (DMSO) and in the presence of the indicated PARP inhibitors ( n = 3, ±SD).
    Figure Legend Snippet: Inhibition of the EGFP gene expression by single apurinic site (AP). ( A ) Vectors used for site-specific insertion of synthetic oligonucleotides containing a single AP lesion (tetrahydrofuran) on the place of dG in the transcribed (TS) or the non-transcribed (NTS) strand of the EGFP gene. ( B ) Activity of APE1 toward plasmid substrates containing single synthetic AP lesion and the effects of the specified phosphorothioate linkages. ( C ) Fluorescence distribution plots of HeLa cells 24 h post-transfection with the expression constructs containing the indicated modifications in the specified DNA strand (TS or NTS), compared to the ‘G’ construct (overlaid blue line). Analogous plots for cells incubated in parallel with PARP inhibitors are shown in Supplementary Figure S5. ( D ) Mean relative EGFP expression of the specified constructs in the absence (DMSO) and in the presence of the indicated PARP inhibitors ( n = 3, ±SD).

    Techniques Used: Inhibition, Expressing, Activity Assay, Plasmid Preparation, Fluorescence, Transfection, Construct, Incubation

    26) Product Images from "Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector"

    Article Title: Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

    Journal: Molecular Therapy

    doi: 10.1038/mt.2015.187

    Cell density transfection optimization experiments and impact of cell culture age on vector production . ( a ) 1 × 10 6 and 2 × 10 6 viable cells/ml were transfected with 1, 1.5, and 2 μg of plasmid DNA
    Figure Legend Snippet: Cell density transfection optimization experiments and impact of cell culture age on vector production . ( a ) 1 × 10 6 and 2 × 10 6 viable cells/ml were transfected with 1, 1.5, and 2 μg of plasmid DNA

    Techniques Used: Transfection, Cell Culture, Plasmid Preparation

    27) Product Images from "Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes"

    Article Title: Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193066

    Excision of H3K4me3/DNaseI signatures by dual guideRNA CRISPR vectors. A) Primary human monocyte DNase- and ChIP-Seq (H3K4me3 and K3K27me3) coverages at the transcriptional start sites of the MALAT1, miR-146a and miR-155 ncRNA genes. Regions selected for targeted excision are marked (dashed lines). B) Genomic PCR with primers flanking the respective H3K4me3/DNaseI element to be excised. Besides the wild-type band (WT) a band of reduced size (ΔTSS) is detected after transfection of HEK293 cells with the respective ncRNA knockout CRISPR construct (KO) but not after transfection of a control CRISPR construct (Ctrl.). Position of the DNA ladder bands (2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp) is indicated to the left of each gel image. C) Sanger-sequencing of ΔTSS bands validates the deletion in the MALAT1, miR155 and miR146a promoter, respectively. Positions of guideRNAs (gRNA) and protospacer-adjacent motifs (PAM) are indicated.
    Figure Legend Snippet: Excision of H3K4me3/DNaseI signatures by dual guideRNA CRISPR vectors. A) Primary human monocyte DNase- and ChIP-Seq (H3K4me3 and K3K27me3) coverages at the transcriptional start sites of the MALAT1, miR-146a and miR-155 ncRNA genes. Regions selected for targeted excision are marked (dashed lines). B) Genomic PCR with primers flanking the respective H3K4me3/DNaseI element to be excised. Besides the wild-type band (WT) a band of reduced size (ΔTSS) is detected after transfection of HEK293 cells with the respective ncRNA knockout CRISPR construct (KO) but not after transfection of a control CRISPR construct (Ctrl.). Position of the DNA ladder bands (2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp) is indicated to the left of each gel image. C) Sanger-sequencing of ΔTSS bands validates the deletion in the MALAT1, miR155 and miR146a promoter, respectively. Positions of guideRNAs (gRNA) and protospacer-adjacent motifs (PAM) are indicated.

    Techniques Used: CRISPR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Knock-Out, Construct, Sequencing

    28) Product Images from "Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis"

    Article Title: Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis

    Journal: Genome Medicine

    doi: 10.1186/s13073-017-0479-0

    Irys/Saphyr chip nanochannel structure and DNA loading. The labeled dsDNA is loaded into two flowcells of either Irys or Saphyr chips. The applied voltage concentrates the coiled DNA at the lip ( left ). Later, DNA is pushed through pillars ( middle ) to uncoil/straighten, then into nanochannels ( right ). DNA is stopped and imaged in the nanochannels. Blue = staining of DNA backbone, green = fluorescently labeled nicked sites
    Figure Legend Snippet: Irys/Saphyr chip nanochannel structure and DNA loading. The labeled dsDNA is loaded into two flowcells of either Irys or Saphyr chips. The applied voltage concentrates the coiled DNA at the lip ( left ). Later, DNA is pushed through pillars ( middle ) to uncoil/straighten, then into nanochannels ( right ). DNA is stopped and imaged in the nanochannels. Blue = staining of DNA backbone, green = fluorescently labeled nicked sites

    Techniques Used: Chromatin Immunoprecipitation, Labeling, Staining

    29) Product Images from "Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors"

    Article Title: Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072407

    BMPRII suppresses ActRIIA-mediated Smad1 activity. PC3-M cells were transfected with BRE 2 -luciferase, Renilla luciferase, and indicated plasmid DNA with or without indicated siRNA. siRNA lanes marked with a hyphen were transfected with non-targeting siRNA. Two days later cells were lysed and luciferase activity was assessed as in Figure 2B . A) Increased BRE 2 -luciferase activity upon silencing BMPRII is mediated by ActRIIA. Neg = non-targeting siRNA. 2A = siActRIIA. 2B = siActRIIB. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. B ) BMPRII-mediated suppression of BRE 2 -luciferase activity is dependent on ActRIIA expression. BMPRII construct abbreviations as in Figure 4 . Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated 3 times (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. C ) BMPRII suppresses signaling from ActRIIA. Data represent mean ± SD from one representative experiment (N = 2 replicates) of three repeated separately (N = 2). *, p
    Figure Legend Snippet: BMPRII suppresses ActRIIA-mediated Smad1 activity. PC3-M cells were transfected with BRE 2 -luciferase, Renilla luciferase, and indicated plasmid DNA with or without indicated siRNA. siRNA lanes marked with a hyphen were transfected with non-targeting siRNA. Two days later cells were lysed and luciferase activity was assessed as in Figure 2B . A) Increased BRE 2 -luciferase activity upon silencing BMPRII is mediated by ActRIIA. Neg = non-targeting siRNA. 2A = siActRIIA. 2B = siActRIIB. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. B ) BMPRII-mediated suppression of BRE 2 -luciferase activity is dependent on ActRIIA expression. BMPRII construct abbreviations as in Figure 4 . Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated 3 times (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. C ) BMPRII suppresses signaling from ActRIIA. Data represent mean ± SD from one representative experiment (N = 2 replicates) of three repeated separately (N = 2). *, p

    Techniques Used: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Construct

    30) Product Images from "Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge"

    Article Title: Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

    Journal: Materials

    doi: 10.3390/ma11071129

    Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.
    Figure Legend Snippet: Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    31) Product Images from "Zika Virus Non-structural Protein 4A Blocks the RLR-MAVS Signaling"

    Article Title: Zika Virus Non-structural Protein 4A Blocks the RLR-MAVS Signaling

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01350

    NS4A interacts with MAVS and blocks RLR binding to MAVS. (A) Co-immunoprecipitation (co-IP) of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and the indicated FLAG-tagged genes using an anti-FLAG monoclonal antibody, followed by immunoblotting (IB). WCE, whole-cell extract. (B) co-IP of the truncated forms of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and FLAG-tagged MAVS mutants using an anti-FLAG monoclonal antibody. co-IP of (C) FLAG-MDA5 or (D) FLAG-ΔRIG-I with endogenous MAVS from HEK293T cells transfected with FLAG-MDA5 or FLAG-ΔRIG-I and Myc vector or increasing amounts of Myc-NS4A. The plasmid DNA molar ratio of Myc- NS4A to FLAG-MDA5 is 1:3; to FLAG-ΔRIG-I are 1:6, 1:3, and 1:2. (E) Immunofluorescent staining of endogenous MAVS and MDA5 in human trophoblasts. Trophoblasts were untreated (Mock) or transfected with either a FLAG vector or FLAG-NS4A for 24 h and then stimulated with 20 μg/ml of heavy molecular weight polyI:C-H for 8 h. MDA5/MAVS was stained with a rabbit anti-MDA5 and mouse anti-MAVS antibody followed by secondary antibodies conjugated with Alexa Fluor 594/488. The nuclei were counter-stained with DAPI. The images were acquired using an inverted Nikon Eclipse Ti fluorescence microscope. The arrows indicate colocalizations of MDA5 and MAVS. In (A–C) actin is a housekeeping protein control. The arrow heads point to non-specific bands. The data shown are representative of three independent experiments.
    Figure Legend Snippet: NS4A interacts with MAVS and blocks RLR binding to MAVS. (A) Co-immunoprecipitation (co-IP) of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and the indicated FLAG-tagged genes using an anti-FLAG monoclonal antibody, followed by immunoblotting (IB). WCE, whole-cell extract. (B) co-IP of the truncated forms of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and FLAG-tagged MAVS mutants using an anti-FLAG monoclonal antibody. co-IP of (C) FLAG-MDA5 or (D) FLAG-ΔRIG-I with endogenous MAVS from HEK293T cells transfected with FLAG-MDA5 or FLAG-ΔRIG-I and Myc vector or increasing amounts of Myc-NS4A. The plasmid DNA molar ratio of Myc- NS4A to FLAG-MDA5 is 1:3; to FLAG-ΔRIG-I are 1:6, 1:3, and 1:2. (E) Immunofluorescent staining of endogenous MAVS and MDA5 in human trophoblasts. Trophoblasts were untreated (Mock) or transfected with either a FLAG vector or FLAG-NS4A for 24 h and then stimulated with 20 μg/ml of heavy molecular weight polyI:C-H for 8 h. MDA5/MAVS was stained with a rabbit anti-MDA5 and mouse anti-MAVS antibody followed by secondary antibodies conjugated with Alexa Fluor 594/488. The nuclei were counter-stained with DAPI. The images were acquired using an inverted Nikon Eclipse Ti fluorescence microscope. The arrows indicate colocalizations of MDA5 and MAVS. In (A–C) actin is a housekeeping protein control. The arrow heads point to non-specific bands. The data shown are representative of three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Staining, Molecular Weight, Fluorescence, Microscopy

    32) Product Images from "Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis"

    Article Title: Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105223

    Molecular analyses of L. incisa clones transformed with a pRbcS450 construct. (A) PCR analysis: gDNA was amplified with BleF and BleR primers yielding a 415-bp fragment. Lanes: (M) DNA ladder; (-) no template, (Ble) ble plasmid control, (wt) negative control (non-transformed cells); five transformed clones. (B) Southern blot analysis: gDNA isolated from both transgenic and non-transgenic cells, as well as plasmid DNA (pRbcS450), were digested with KpnI restriction enzyme. The blot was hybridized with a probe derived from a 415-bp amplified fragment of the ble gene. Lanes: (M) 1 Kb ladder; (plasmid) positive control; five transformed clones; (wt) negative control (non-transformed cells).
    Figure Legend Snippet: Molecular analyses of L. incisa clones transformed with a pRbcS450 construct. (A) PCR analysis: gDNA was amplified with BleF and BleR primers yielding a 415-bp fragment. Lanes: (M) DNA ladder; (-) no template, (Ble) ble plasmid control, (wt) negative control (non-transformed cells); five transformed clones. (B) Southern blot analysis: gDNA isolated from both transgenic and non-transgenic cells, as well as plasmid DNA (pRbcS450), were digested with KpnI restriction enzyme. The blot was hybridized with a probe derived from a 415-bp amplified fragment of the ble gene. Lanes: (M) 1 Kb ladder; (plasmid) positive control; five transformed clones; (wt) negative control (non-transformed cells).

    Techniques Used: Clone Assay, Transformation Assay, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Negative Control, Southern Blot, Isolation, Transgenic Assay, Derivative Assay, Positive Control

    33) Product Images from "Addressing the Instability of DNA Nanostructures in Tissue Culture"

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture

    Journal: ACS Nano

    doi: 10.1021/nn503513p

    DNA nanostructure sensitivity to cation depletion in tissue culture medium. The three test nanostructures are (a) DNA nano-octahedron (DNO), (b) six-helix bundle nanotube (NT), and (c) 24-helix nanorod (NR). (d–f) Three nanostructures were incubated for 24 h at 37 °C in unmodified RPMI tissue culture medium containing 0.4 mM Mg 2+ or adjusted to 0.7–10 mM Mg 2+ , and the products were analyzed by agarose gel electrophoresis (AGE). In comparison to the control lanes of stable sample, migration of the DNO and NR is retarded after low Mg 2+ incubation, indicative of denaturation, whereas no obvious difference in migration is observed with the NT. (g–i) Transmission electron microscopy images of nanostructures incubated in unmodified medium, showing varying levels of denaturation. (j–l) With RPMI medium adjusted to 6 mM Mg 2+ , structural integrity is maintained in all three designs. M = molecular weight ladder, C = nanostructure in TE + 10 mM Mg 2+ , S = M13 scaffold. Scale bar = 100 nm.
    Figure Legend Snippet: DNA nanostructure sensitivity to cation depletion in tissue culture medium. The three test nanostructures are (a) DNA nano-octahedron (DNO), (b) six-helix bundle nanotube (NT), and (c) 24-helix nanorod (NR). (d–f) Three nanostructures were incubated for 24 h at 37 °C in unmodified RPMI tissue culture medium containing 0.4 mM Mg 2+ or adjusted to 0.7–10 mM Mg 2+ , and the products were analyzed by agarose gel electrophoresis (AGE). In comparison to the control lanes of stable sample, migration of the DNO and NR is retarded after low Mg 2+ incubation, indicative of denaturation, whereas no obvious difference in migration is observed with the NT. (g–i) Transmission electron microscopy images of nanostructures incubated in unmodified medium, showing varying levels of denaturation. (j–l) With RPMI medium adjusted to 6 mM Mg 2+ , structural integrity is maintained in all three designs. M = molecular weight ladder, C = nanostructure in TE + 10 mM Mg 2+ , S = M13 scaffold. Scale bar = 100 nm.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Migration, Transmission Assay, Electron Microscopy, Molecular Weight

    34) Product Images from "Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit"

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    Journal: Scientific Reports

    doi: 10.1038/srep41851

    Detection of pseudo-ccc DNA. ( a ) Schematic representation of the HBV expression unit on the AdVs. Ax-CM103G-kS harbours a replicate-competent mutant HBV genome. Crosses represent silent mutation of HBV S protein. Deleted region of the mutant HBV genome “dP” on Ax-CM103G-dP and Ax-CM114-dP are shown as a grey box. Bold and broken lines represent the ORFs of HBV proteins and non-functional region, respectively. Right-angled arrow, transcription initiation site; DR II/I, direct repeat sequences of HBV genome; HR, homologous region. The other representations are the same as in Figs 1 and 2 . ( b ) PCR detection of the circular HBV genome, AdV genome and GAPDH. HepG2 cells were infected with the indicated AdVs. Mock, mock infection of HepG2 cells. Full-length blots are presented in Supplementary Fig. S9 .
    Figure Legend Snippet: Detection of pseudo-ccc DNA. ( a ) Schematic representation of the HBV expression unit on the AdVs. Ax-CM103G-kS harbours a replicate-competent mutant HBV genome. Crosses represent silent mutation of HBV S protein. Deleted region of the mutant HBV genome “dP” on Ax-CM103G-dP and Ax-CM114-dP are shown as a grey box. Bold and broken lines represent the ORFs of HBV proteins and non-functional region, respectively. Right-angled arrow, transcription initiation site; DR II/I, direct repeat sequences of HBV genome; HR, homologous region. The other representations are the same as in Figs 1 and 2 . ( b ) PCR detection of the circular HBV genome, AdV genome and GAPDH. HepG2 cells were infected with the indicated AdVs. Mock, mock infection of HepG2 cells. Full-length blots are presented in Supplementary Fig. S9 .

    Techniques Used: Countercurrent Chromatography, Expressing, Mutagenesis, Functional Assay, Polymerase Chain Reaction, Infection

    35) Product Images from "Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans"

    Article Title: Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00671

    Released DNA in response to human monocytes traps Candida albicans . (A) DNA released by monocytes forms traps and immobilizes C. albicans , whereas whole glucan particles (WGP) or LPS have no effect. Blue: C. albicans , green: SYTOX green. (B) DNAse treatment leads to digestion and disappearance of DNA traps. Green: SYTOX green. Pictures in (A,B) were taken with an LSM710 microscope (Zeiss), fitted with 20× 1.4 NA lens or 40× 1.4 NA oil-immersion lens or 63× 1.4 NA oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Scale bar: 10 µm. Scanning electron microscopy of (C) blood monocytes alone after 4 h. Scale bar: 10 µm. (D,E) DNA fibers and nets that trap C. albicans cells and also hyphae. Scale bar: 10 µm. (F) Monocytic extracellular traps (MoETs) form dense structures that cover hyphae. Scale bar: 1 µm. (G) MoETs are covered with proteins and form strings with bead-like structures. Scale bar: 200 nm. (H) Magnification of MoETs demonstrates a dense network that covers C. albicans and damages the surface. Scale bars = 10 μm, 1 µm, and 200 nm.
    Figure Legend Snippet: Released DNA in response to human monocytes traps Candida albicans . (A) DNA released by monocytes forms traps and immobilizes C. albicans , whereas whole glucan particles (WGP) or LPS have no effect. Blue: C. albicans , green: SYTOX green. (B) DNAse treatment leads to digestion and disappearance of DNA traps. Green: SYTOX green. Pictures in (A,B) were taken with an LSM710 microscope (Zeiss), fitted with 20× 1.4 NA lens or 40× 1.4 NA oil-immersion lens or 63× 1.4 NA oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Scale bar: 10 µm. Scanning electron microscopy of (C) blood monocytes alone after 4 h. Scale bar: 10 µm. (D,E) DNA fibers and nets that trap C. albicans cells and also hyphae. Scale bar: 10 µm. (F) Monocytic extracellular traps (MoETs) form dense structures that cover hyphae. Scale bar: 1 µm. (G) MoETs are covered with proteins and form strings with bead-like structures. Scale bar: 200 nm. (H) Magnification of MoETs demonstrates a dense network that covers C. albicans and damages the surface. Scale bars = 10 μm, 1 µm, and 200 nm.

    Techniques Used: Microscopy, Software, Electron Microscopy

    Monocytic extracellular traps stain positive for CD14, CD115, and Ly6c . Extracellular traps derived from monocytes contain the monocytic marker (A) CD14. Human blood monocytes were incubated with Candida albicans (1:1) in growth medium with 10% NHS for 4 h, fixed, and stained with specific antibodies. Orange: SYTOX orange, green: CD14. (B) Mouse monocytes form extracellular traps (arrows) in mouse liver sections derived from C. albicans -infected mice. The traps show the monocytic markers CD115 and Ly6c, but not Ly6g, a marker for neutrophils. Representative stainings from at least three independent experiments are shown. Orange: DNA (SYTOX orange), green: CD115, red: Ly6c, blue: Ly6g. Pictures were taken with an LSM710 microscope (Zeiss), with 40× 1.4 NA oil-immersion lens or 100× 1.4 NA oil-immersion lens, and processed, using the ZEN 2011 software (Zeiss). Scale bar: 10 µm.
    Figure Legend Snippet: Monocytic extracellular traps stain positive for CD14, CD115, and Ly6c . Extracellular traps derived from monocytes contain the monocytic marker (A) CD14. Human blood monocytes were incubated with Candida albicans (1:1) in growth medium with 10% NHS for 4 h, fixed, and stained with specific antibodies. Orange: SYTOX orange, green: CD14. (B) Mouse monocytes form extracellular traps (arrows) in mouse liver sections derived from C. albicans -infected mice. The traps show the monocytic markers CD115 and Ly6c, but not Ly6g, a marker for neutrophils. Representative stainings from at least three independent experiments are shown. Orange: DNA (SYTOX orange), green: CD115, red: Ly6c, blue: Ly6g. Pictures were taken with an LSM710 microscope (Zeiss), with 40× 1.4 NA oil-immersion lens or 100× 1.4 NA oil-immersion lens, and processed, using the ZEN 2011 software (Zeiss). Scale bar: 10 µm.

    Techniques Used: Staining, Derivative Assay, Marker, Incubation, Infection, Mouse Assay, Microscopy, Software

    Human blood-derived monocytes release DNA in response to Candida albicans . (A) Human blood monocytes (red) spontaneously engulf GFP- C. albicans cells (green). Representative cells from three independent experiments are shown. Pictures were taken with a LSM710 microscope (Zeiss), fitted with a 40×, 1.4 NA, oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Scale bar: 10 µm. (B) Representative example of gating strategy to determine by flow cytometry single- and double-positive monocytes (GFP and DID). (C) Phagocytosis of GFP- C. albicans is enhanced in complement active human serum (NHS). GFP-expressing C. albicans were incubated with monocytes in the absence or presence of human serum and the phagocytosis rate was determined by flow cytometry. Data represent mean values ± SDs of four independent experiments (Phagocytosis in NHS versus iNHS after 1 h ** p = 0.0083; one sided Student’s t -test). (D) Peripheral blood monocytes react on GFP- C. albicans by the release of DNA, which was stained with SYTOX blue (upper panel). Release of DNA increased over time (lower panel) as measured by ZEN 2011 software (Zeiss). (E) Human blood monocytes phagocytose C. albicans cells, decondense after about 150 min the chromosomal DNA, and subsequently burst out the DNA. Blood monocytes were incubated with GFP- C. albicans cells at a ratio of 1:1 in the presence of 10% complement active human serum and interaction was followed by life time imaging using LSM (see also Video S1 in Supplementary Material). Red: DiD stained membrane, blue: SYTOX blue, green: GFP- Candida . (F) Both types of monocytes CD14 ++ CD16 − and CD14 + CD16 + release DNA in response to C. albicans at about 150 min. Green: CD14, red: CD16, blue: SYTOX blue. Representative cells from at least three independent experiments. Pictures were taken with an LSM710 microscope (Zeiss), fitted with a 40×, 1.4 NA, oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Size bars: 10 µm.
    Figure Legend Snippet: Human blood-derived monocytes release DNA in response to Candida albicans . (A) Human blood monocytes (red) spontaneously engulf GFP- C. albicans cells (green). Representative cells from three independent experiments are shown. Pictures were taken with a LSM710 microscope (Zeiss), fitted with a 40×, 1.4 NA, oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Scale bar: 10 µm. (B) Representative example of gating strategy to determine by flow cytometry single- and double-positive monocytes (GFP and DID). (C) Phagocytosis of GFP- C. albicans is enhanced in complement active human serum (NHS). GFP-expressing C. albicans were incubated with monocytes in the absence or presence of human serum and the phagocytosis rate was determined by flow cytometry. Data represent mean values ± SDs of four independent experiments (Phagocytosis in NHS versus iNHS after 1 h ** p = 0.0083; one sided Student’s t -test). (D) Peripheral blood monocytes react on GFP- C. albicans by the release of DNA, which was stained with SYTOX blue (upper panel). Release of DNA increased over time (lower panel) as measured by ZEN 2011 software (Zeiss). (E) Human blood monocytes phagocytose C. albicans cells, decondense after about 150 min the chromosomal DNA, and subsequently burst out the DNA. Blood monocytes were incubated with GFP- C. albicans cells at a ratio of 1:1 in the presence of 10% complement active human serum and interaction was followed by life time imaging using LSM (see also Video S1 in Supplementary Material). Red: DiD stained membrane, blue: SYTOX blue, green: GFP- Candida . (F) Both types of monocytes CD14 ++ CD16 − and CD14 + CD16 + release DNA in response to C. albicans at about 150 min. Green: CD14, red: CD16, blue: SYTOX blue. Representative cells from at least three independent experiments. Pictures were taken with an LSM710 microscope (Zeiss), fitted with a 40×, 1.4 NA, oil-immersion lens, and processed, using ZEN 2011 software (Zeiss). Size bars: 10 µm.

    Techniques Used: Derivative Assay, Microscopy, Software, Flow Cytometry, Cytometry, Expressing, Incubation, Staining, Imaging

    36) Product Images from "Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis"

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.02119-16

    Permissive and nonpermissive human cells support FeLV-B entry and early proviral DNA synthesis at similar levels. (A) Initial studies using standard PCR with primers based on FeLV LTR or envelope sequences showed marked increases in proviral DNA levels between 0.5 and 6 h after the exposure of cells to FeLV-B at an MOI of 1. Because no PIT-1-negative cell lines were identified, a receptor-negative control was generated for this assay by comparing the FeLV-A infection of permissive feline cells (3201 cells) and of Reh cells, which lack detectable expression of mRNA for the human homologue of FeLV-A receptor THTR1. Loading controls used human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pan-Myc primers, which efficiently detect human and feline DNA. (B) A wider analysis of FeLV-B infection of human hematopoietic cell lines. Proviral DNA levels were analyzed by quantitative PCR. The line graphs show changes in FeLV DNA concentrations (detected by EnvB primers) relative to those of a housekeeping gene control (β2-microglobulin) between 0.5 and 6 h (top) or between 6 h and 14 days (bottom) after infection. The y axis shows the threshold cycle ( C T ) value relative to that of the β2-microglobulin control (taken as zero). Partially permissive cells were CEM, Jurkat, and Raji cells. Nonpermissive cells were PBMCs ( n = 2), Reh cells, and ALL/MIK cells. Lymphoblastoid cell lines were LCL30, LCL98, LCL113, and LCL114.
    Figure Legend Snippet: Permissive and nonpermissive human cells support FeLV-B entry and early proviral DNA synthesis at similar levels. (A) Initial studies using standard PCR with primers based on FeLV LTR or envelope sequences showed marked increases in proviral DNA levels between 0.5 and 6 h after the exposure of cells to FeLV-B at an MOI of 1. Because no PIT-1-negative cell lines were identified, a receptor-negative control was generated for this assay by comparing the FeLV-A infection of permissive feline cells (3201 cells) and of Reh cells, which lack detectable expression of mRNA for the human homologue of FeLV-A receptor THTR1. Loading controls used human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pan-Myc primers, which efficiently detect human and feline DNA. (B) A wider analysis of FeLV-B infection of human hematopoietic cell lines. Proviral DNA levels were analyzed by quantitative PCR. The line graphs show changes in FeLV DNA concentrations (detected by EnvB primers) relative to those of a housekeeping gene control (β2-microglobulin) between 0.5 and 6 h (top) or between 6 h and 14 days (bottom) after infection. The y axis shows the threshold cycle ( C T ) value relative to that of the β2-microglobulin control (taken as zero). Partially permissive cells were CEM, Jurkat, and Raji cells. Nonpermissive cells were PBMCs ( n = 2), Reh cells, and ALL/MIK cells. Lymphoblastoid cell lines were LCL30, LCL98, LCL113, and LCL114.

    Techniques Used: DNA Synthesis, Polymerase Chain Reaction, Negative Control, Generated, Infection, Expressing, Real-time Polymerase Chain Reaction

    Persistence of integrated FeLV-B DNA in human PBMCs. (A) Semiquantitative PCR reveals dose-dependent persistence of FeLV DNA in human PBMCs at 14 days postinfection. Fully permissive HEK293 control cells show stable levels by 3 days postinfection. (B) Results of a quantitative PCR time course analysis for FeLV sequences in PBMC DNA, with and without a preamplification step using primers to FeLV Gag and consensus human Alu sequences. The threshold cycle ( C T ) value is inversely proportional to the DNA content. (C) Results of semiquantitative PCR for LTR circles in FeLV-B-infected cells 3 days (PBMC, Reh, and KYO-1 cells) or 24 h (3201 and HEK293 cells) after infection. While single and double LTR circle forms are readily detected in productively infected HEK293 and 3201 cells, they are barely detectable in Reh cells and PBMCs relative to levels in permissive HEK293 control cells. The loading control was provided by PCR amplification with conserved Myc primers (pan-Myc).
    Figure Legend Snippet: Persistence of integrated FeLV-B DNA in human PBMCs. (A) Semiquantitative PCR reveals dose-dependent persistence of FeLV DNA in human PBMCs at 14 days postinfection. Fully permissive HEK293 control cells show stable levels by 3 days postinfection. (B) Results of a quantitative PCR time course analysis for FeLV sequences in PBMC DNA, with and without a preamplification step using primers to FeLV Gag and consensus human Alu sequences. The threshold cycle ( C T ) value is inversely proportional to the DNA content. (C) Results of semiquantitative PCR for LTR circles in FeLV-B-infected cells 3 days (PBMC, Reh, and KYO-1 cells) or 24 h (3201 and HEK293 cells) after infection. While single and double LTR circle forms are readily detected in productively infected HEK293 and 3201 cells, they are barely detectable in Reh cells and PBMCs relative to levels in permissive HEK293 control cells. The loading control was provided by PCR amplification with conserved Myc primers (pan-Myc).

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Infection, Amplification

    Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.
    Figure Legend Snippet: Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Techniques Used: Expressing, Genomic Sequencing, Clone Assay, Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Sequencing, Mutagenesis

    37) Product Images from "Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1"

    Article Title: Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1

    Journal: Nature

    doi: 10.1038/nature21678

    Nuclear-targeted increases of UBE3A repress ASD network gene Cbln1 that is required in VGluT2 neurons for sociability a , Protein interaction sub-cluster enriched for glutamatergic synapse proteins encoded by autism-linked 13 and Ube3a up- and down-regulated genes. b , q-RT-PCR validates Cbln1 in Ube3a1x 4 , Ube3aNLS, Ube3a2x 4 , m-Ube3a mKO 21 (n=3 pooled samples of two mice each), Ube3a2x (untagged) and p-Ube3a mKO 5 (n=6–12/group). Transgene constructs or breeding strategy schematic shown (F: FLAG-tags, NLS: nuclear localization signal). c , q-RT-PCR validates Cbln3 ( Cerebellin-3), Grid-2 (glutamate receptor ionotropic delta subunit-2), Homer-3, Inadl (InaD-like), Grm4 (Glutamate receptor metabotropic 4), En2 (engrailed-2), Gabra6 (GABA-A subunit alpha 6), Il16 (interleukin-16), Fgf7 (fibroblast growth factor 7), Chd7 (chomodomain helicase DNA binding protein 7), Atp2a3 (ATPase Calcium transporting ubiquitous), Tfap2b (transcription factor AP-2 beta), Calb2 (Calbindin 2), Gdf10 (growth differentiation factor 10), Zfp521 (Zinc finger protein 521), Ebf1 (early B-cell factor 1). d , Sociability of female mutant/transgenic and WT littermate mice. e , USVs during genotype-matched pairings. f , qRT-PCR for Cbln1 (Cblm: cerebellum, MA: medial amygdala, HPc: hippocampus, ECx: entorhinal cortex, VTA: ventral tegmental area, RSCx: retrosplenial cortex, VMH: ventromedial hypothalamus); g , sociability (Cre- n=8, Cre+ n=10); and h , USVs in male-female (Cre- n=14, Cre+ n=8) and genotype-matched female-female pairs (Cre- n=11, Cre+ n=16). Mean ± SEM plotted. For three-chamber experiments, “sniffing time” analyzed by two-way repeated-measures ANOVA. F statistics and p values for “interaction effects” between chamber side (social versus opposite) or experimental group (viruses, treatments or genotypes) and “main effects” in Supplementary Table 5 . For significant interaction and/or main effects , a Bonferroni post-hoc test p-value is reported (adjusted for multiple comparisons, corresponding to * or ns [not significant] designations).
    Figure Legend Snippet: Nuclear-targeted increases of UBE3A repress ASD network gene Cbln1 that is required in VGluT2 neurons for sociability a , Protein interaction sub-cluster enriched for glutamatergic synapse proteins encoded by autism-linked 13 and Ube3a up- and down-regulated genes. b , q-RT-PCR validates Cbln1 in Ube3a1x 4 , Ube3aNLS, Ube3a2x 4 , m-Ube3a mKO 21 (n=3 pooled samples of two mice each), Ube3a2x (untagged) and p-Ube3a mKO 5 (n=6–12/group). Transgene constructs or breeding strategy schematic shown (F: FLAG-tags, NLS: nuclear localization signal). c , q-RT-PCR validates Cbln3 ( Cerebellin-3), Grid-2 (glutamate receptor ionotropic delta subunit-2), Homer-3, Inadl (InaD-like), Grm4 (Glutamate receptor metabotropic 4), En2 (engrailed-2), Gabra6 (GABA-A subunit alpha 6), Il16 (interleukin-16), Fgf7 (fibroblast growth factor 7), Chd7 (chomodomain helicase DNA binding protein 7), Atp2a3 (ATPase Calcium transporting ubiquitous), Tfap2b (transcription factor AP-2 beta), Calb2 (Calbindin 2), Gdf10 (growth differentiation factor 10), Zfp521 (Zinc finger protein 521), Ebf1 (early B-cell factor 1). d , Sociability of female mutant/transgenic and WT littermate mice. e , USVs during genotype-matched pairings. f , qRT-PCR for Cbln1 (Cblm: cerebellum, MA: medial amygdala, HPc: hippocampus, ECx: entorhinal cortex, VTA: ventral tegmental area, RSCx: retrosplenial cortex, VMH: ventromedial hypothalamus); g , sociability (Cre- n=8, Cre+ n=10); and h , USVs in male-female (Cre- n=14, Cre+ n=8) and genotype-matched female-female pairs (Cre- n=11, Cre+ n=16). Mean ± SEM plotted. For three-chamber experiments, “sniffing time” analyzed by two-way repeated-measures ANOVA. F statistics and p values for “interaction effects” between chamber side (social versus opposite) or experimental group (viruses, treatments or genotypes) and “main effects” in Supplementary Table 5 . For significant interaction and/or main effects , a Bonferroni post-hoc test p-value is reported (adjusted for multiple comparisons, corresponding to * or ns [not significant] designations).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Construct, Binding Assay, Mutagenesis, Transgenic Assay, Quantitative RT-PCR

    38) Product Images from "A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells"

    Article Title: A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182941

    Cellular toxicity can be determined in described flow cytometric method that quantifies transfection efficiency without the use of a viability dye. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and the TransITX2 transfection reagent as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and three independent times with specific (un)labelled nucleic acids. Median and interquartile range (IQR) are shown. The non-parametric statistical Kruskal-Wallis test was used for comparisons between groups. Quantification of cell death by forward and side scatter parameters, and doublet discrimination [18.4% (8.6)] gave similar results compared to quantification of cell death by nucleic acid viability dye (7-AAD) [20.50% (11.7)] and amine viability dye [24.4 (11.1)](p = 0.379). Of note use of viability cell dye cannot be used in electroporation experiments (e.g. Jurkat E6 cells electroporation with FITC-labeled DNA mCherry plasmid) due to the mechanisms of action of electroporation methods and dye exclusion tests for cell viability dyes.
    Figure Legend Snippet: Cellular toxicity can be determined in described flow cytometric method that quantifies transfection efficiency without the use of a viability dye. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and the TransITX2 transfection reagent as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and three independent times with specific (un)labelled nucleic acids. Median and interquartile range (IQR) are shown. The non-parametric statistical Kruskal-Wallis test was used for comparisons between groups. Quantification of cell death by forward and side scatter parameters, and doublet discrimination [18.4% (8.6)] gave similar results compared to quantification of cell death by nucleic acid viability dye (7-AAD) [20.50% (11.7)] and amine viability dye [24.4 (11.1)](p = 0.379). Of note use of viability cell dye cannot be used in electroporation experiments (e.g. Jurkat E6 cells electroporation with FITC-labeled DNA mCherry plasmid) due to the mechanisms of action of electroporation methods and dye exclusion tests for cell viability dyes.

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Labeling, Electroporation, Plasmid Preparation

    Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.
    Figure Legend Snippet: Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Labeling, Plasmid Preparation, MANN-WHITNEY

    Determination of cell viability in described flow cytometric method that quantifies transfection efficiency. Representative transfections are shown. 293T cells underwent chemical transfection using the TransITX2 transfection reagent and the mCherry plasmid as described in Fig 1 . Cell viability and co-expression of DNA taken up by cells and target protein were analyzed 24 h after transfection. Cell viability was assessed by flow cytometry using two independent approaches: a) viable cells were gated based on forward and side scatter as shown in Fig 1 b) viable cells were gated based on nucleic acid [7-AAD: 7-Aminoactinomycin D, A-C] and amine reactive (Ghost 780 Live/dead dye, D-F) viability dyes. Representative plots from 3 independent experiments are shown. As expected the chemical transfection and the DNA plasmids per se are toxic to the cells. Similar data were obtained using 2 independent viability dyes [7AAD (A), Ghost 780 (D)]. Overall cellular toxicity was similar (10–40%) among the 3 methods [FSC/SSC ( Fig 1 ), nucleic acid and amine reactive death dyes]( Fig 4 ). The mean viability of the untransfected 293T cells was > 85%. In addition, determination of the transfection efficiency based on two independent readouts (DNA plasmid uptake and protein expression) was similar by gating on viable cells based on either FSC/SSC (B, E) or viability dye (C, F).
    Figure Legend Snippet: Determination of cell viability in described flow cytometric method that quantifies transfection efficiency. Representative transfections are shown. 293T cells underwent chemical transfection using the TransITX2 transfection reagent and the mCherry plasmid as described in Fig 1 . Cell viability and co-expression of DNA taken up by cells and target protein were analyzed 24 h after transfection. Cell viability was assessed by flow cytometry using two independent approaches: a) viable cells were gated based on forward and side scatter as shown in Fig 1 b) viable cells were gated based on nucleic acid [7-AAD: 7-Aminoactinomycin D, A-C] and amine reactive (Ghost 780 Live/dead dye, D-F) viability dyes. Representative plots from 3 independent experiments are shown. As expected the chemical transfection and the DNA plasmids per se are toxic to the cells. Similar data were obtained using 2 independent viability dyes [7AAD (A), Ghost 780 (D)]. Overall cellular toxicity was similar (10–40%) among the 3 methods [FSC/SSC ( Fig 1 ), nucleic acid and amine reactive death dyes]( Fig 4 ). The mean viability of the untransfected 293T cells was > 85%. In addition, determination of the transfection efficiency based on two independent readouts (DNA plasmid uptake and protein expression) was similar by gating on viable cells based on either FSC/SSC (B, E) or viability dye (C, F).

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Expressing

    Labeling of the transfected nucleic acids did not affect their function. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and the TransITX2 transfection reagent as described in Methods. Jurkat E6 cells underwent electroporation with FITC-labeled DNA mCherry plasmid as described in Methods. Each experiment was performed in triplicates and three independent times with specific (un)labelled nucleic acids. Viable cells were gated based on either forward and side scatter (FSC/SSC) or viability dye as shown in Fig 3 and transfection efficiency was determined by comparing protein expression compared to untransfected control. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (chemical transfection versus electroporation in 293T cells versus Jurkat E6 cells). The non-parametric statistical Kruskal-Wallis test was used for comparisons between the labeled and unlabeled transfected nucleic acids. Median and interquartile range (IQR) are shown. The use of unlabeled transfected nucleic acids (standard transfection method) lead to similar protein expression of target gene [100, (14)] compared to the labeled transfected nucleic acids, regardless of the method of viability gating (FSC/SSC; 94.5, (20) vs viability dye; 101 (16)] (p = 0.745).
    Figure Legend Snippet: Labeling of the transfected nucleic acids did not affect their function. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and the TransITX2 transfection reagent as described in Methods. Jurkat E6 cells underwent electroporation with FITC-labeled DNA mCherry plasmid as described in Methods. Each experiment was performed in triplicates and three independent times with specific (un)labelled nucleic acids. Viable cells were gated based on either forward and side scatter (FSC/SSC) or viability dye as shown in Fig 3 and transfection efficiency was determined by comparing protein expression compared to untransfected control. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (chemical transfection versus electroporation in 293T cells versus Jurkat E6 cells). The non-parametric statistical Kruskal-Wallis test was used for comparisons between the labeled and unlabeled transfected nucleic acids. Median and interquartile range (IQR) are shown. The use of unlabeled transfected nucleic acids (standard transfection method) lead to similar protein expression of target gene [100, (14)] compared to the labeled transfected nucleic acids, regardless of the method of viability gating (FSC/SSC; 94.5, (20) vs viability dye; 101 (16)] (p = 0.745).

    Techniques Used: Labeling, Transfection, Electroporation, Plasmid Preparation, Expressing

    Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using different commercially available transfection reagents and the mCherry plasmid (plasmid A; A, B, C, D) or the NL4.3 plasmid (plasmid B; E, F, G, H) as described in Fig 1 . Co-expression of DNA taken up by cells and target protein were analyzed at 24 hours after transfection. Data are means of triplicates from three independent experiments. For each reagent the ratio of transfection reagent to DNA amount was optimized as per manufacturer`s instructions and the same (1 μg) amount of DNA was used.
    Figure Legend Snippet: Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using different commercially available transfection reagents and the mCherry plasmid (plasmid A; A, B, C, D) or the NL4.3 plasmid (plasmid B; E, F, G, H) as described in Fig 1 . Co-expression of DNA taken up by cells and target protein were analyzed at 24 hours after transfection. Data are means of triplicates from three independent experiments. For each reagent the ratio of transfection reagent to DNA amount was optimized as per manufacturer`s instructions and the same (1 μg) amount of DNA was used.

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Expressing

    Flow cytometric analysis of DNA uptake and protein expression over time in described flow cytometric method that quantifies transfection efficiency. 293T cells (
    Figure Legend Snippet: Flow cytometric analysis of DNA uptake and protein expression over time in described flow cytometric method that quantifies transfection efficiency. 293T cells (

    Techniques Used: Flow Cytometry, Expressing, Cytometry, Transfection

    Flow cytometric determination of transfection efficiency based on two independent readouts (DNA plasmid uptake and protein expression). Representative transfections are shown. 293T cells underwent chemical transfection using the TransITX2 transfection reagent as described in Methods. The same amount (1 μg) of DNA was used for two independent plasmids: a small (B: pUltraHot expressing mCherry, 8.3 kb) and a large (C: pNL4-3 expressing p24, 14.0 kb) DNA plasmid. Gating strategy is shown: A) forward and side scatter B) discrimination of doublets C, D) two independent readouts of transfection efficiency. FITC fluorescence corresponds to the uptake of FITC-labeled plasmid DNA (y-axis). A fluorochrome that has no spectral overlap with FITC is used to quantify protein expression. Either a fluorescent protein can be used (e.g. mCherry; shown in C) or a protein labeled with a fluorescent-labeled antibody (e.g. intracellular expression of HIV-1 p24 protein was detected by an CF647-labeled anti-p24 antibody; shown in D). Co-expression of DNA taken up by cells and target protein were analyzed 24 h after transfection. The numbers in the quadrants indicate the percentages of viable cells that took up the FITC labeled DNA plasmid versus the expressed protein that was detected. The following dot plots are shown for each chemical transfection in 293T cells: i) untransfected cells (negative control), ii) cells transfected with FITC-labeled DNA plasmid harvested before protein expression occurred (3 hours post transfection), iii) cells transfected with unlabeled plasmid harvested 24 hours after transfection (when protein expression can be quantified) iv) cells transfected with FITC-labeled DNA plasmid and harvested 24 hours after transfection (when protein expression can be quantified). In this plot Q3 quadrant demonstrates many cells that express protein but do not show any fluorescence associated with uptake of the plasmid DNA. This may reflect effects of the cellular machinery on FITC fluorescence (see Discussion ). Either Q1+Q2 (DNA signal) or Q2+Q3 (protein signal) should be used as readouts of transfection efficiency. E. Transfection efficiency was quantified in human lymphocytes (Jurkat E6 cells) harvested 24 hours after electroporation with FITC-labeled DNA mCherry plasmid without the need to use co-transfection of 2 different plasmids and GFP reporter.
    Figure Legend Snippet: Flow cytometric determination of transfection efficiency based on two independent readouts (DNA plasmid uptake and protein expression). Representative transfections are shown. 293T cells underwent chemical transfection using the TransITX2 transfection reagent as described in Methods. The same amount (1 μg) of DNA was used for two independent plasmids: a small (B: pUltraHot expressing mCherry, 8.3 kb) and a large (C: pNL4-3 expressing p24, 14.0 kb) DNA plasmid. Gating strategy is shown: A) forward and side scatter B) discrimination of doublets C, D) two independent readouts of transfection efficiency. FITC fluorescence corresponds to the uptake of FITC-labeled plasmid DNA (y-axis). A fluorochrome that has no spectral overlap with FITC is used to quantify protein expression. Either a fluorescent protein can be used (e.g. mCherry; shown in C) or a protein labeled with a fluorescent-labeled antibody (e.g. intracellular expression of HIV-1 p24 protein was detected by an CF647-labeled anti-p24 antibody; shown in D). Co-expression of DNA taken up by cells and target protein were analyzed 24 h after transfection. The numbers in the quadrants indicate the percentages of viable cells that took up the FITC labeled DNA plasmid versus the expressed protein that was detected. The following dot plots are shown for each chemical transfection in 293T cells: i) untransfected cells (negative control), ii) cells transfected with FITC-labeled DNA plasmid harvested before protein expression occurred (3 hours post transfection), iii) cells transfected with unlabeled plasmid harvested 24 hours after transfection (when protein expression can be quantified) iv) cells transfected with FITC-labeled DNA plasmid and harvested 24 hours after transfection (when protein expression can be quantified). In this plot Q3 quadrant demonstrates many cells that express protein but do not show any fluorescence associated with uptake of the plasmid DNA. This may reflect effects of the cellular machinery on FITC fluorescence (see Discussion ). Either Q1+Q2 (DNA signal) or Q2+Q3 (protein signal) should be used as readouts of transfection efficiency. E. Transfection efficiency was quantified in human lymphocytes (Jurkat E6 cells) harvested 24 hours after electroporation with FITC-labeled DNA mCherry plasmid without the need to use co-transfection of 2 different plasmids and GFP reporter.

    Techniques Used: Flow Cytometry, Transfection, Plasmid Preparation, Expressing, Fluorescence, Labeling, Negative Control, Electroporation, Cotransfection

    39) Product Images from "The Lyme Disease Pathogen Borrelia burgdorferi Infects Murine Bone and Induces Trabecular Bone Loss"

    Article Title: The Lyme Disease Pathogen Borrelia burgdorferi Infects Murine Bone and Induces Trabecular Bone Loss

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00781-16

    Detection of B. burgdorferi DNA in bones. Quantitative real-time PCR measurement of copies of the B. burgdorferi flaB DNA sequence in distal tibial halves and L6 vertebrae from 12-week-old male C3H/HeN mice 4 weeks after inoculation with 10 4 B. burgdorferi cells or vehicle alone (mock). Shown are median and individual numbers of flaB copies per 1,000 copies of the mouse nidogen gene ( nido ) in each sample. n = 14 to 15 per group. Statistical analysis used a two-way analysis of variance with the Holm-Sidak posttest of log-transformed values. *, P
    Figure Legend Snippet: Detection of B. burgdorferi DNA in bones. Quantitative real-time PCR measurement of copies of the B. burgdorferi flaB DNA sequence in distal tibial halves and L6 vertebrae from 12-week-old male C3H/HeN mice 4 weeks after inoculation with 10 4 B. burgdorferi cells or vehicle alone (mock). Shown are median and individual numbers of flaB copies per 1,000 copies of the mouse nidogen gene ( nido ) in each sample. n = 14 to 15 per group. Statistical analysis used a two-way analysis of variance with the Holm-Sidak posttest of log-transformed values. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Mouse Assay, Transformation Assay

    40) Product Images from "A Novel Slug-containing Negative Feedback Loop regulates SCF/c-Kit-Mediated Hematopoietic Stem Cell Self-renewal"

    Article Title: A Novel Slug-containing Negative Feedback Loop regulates SCF/c-Kit-Mediated Hematopoietic Stem Cell Self-renewal

    Journal: Leukemia

    doi: 10.1038/leu.2016.201

    Key Role of c-Myc and FoxM1 in SCF/c-Kit-Slug Feedback Loop (A) Analysis of c-Myc and FoxM1 occupancies at the Slug promoter by ChIP analysis. Primary BM cells were transduced with retroviral particles containing c-Myc and FoxM1, respectively. The genomic DNA was pulled down by anti-Flag and IgG control antibodies, and amplified with primer pairs F1/R1 (−856 to −691bp), F2/R2 (−1389 to −1234bp), F3/R3 (−1772 to −1605bp), F4/R4 (−597 to −410bp) F5/R5 (−1203 to −1012bp), F6/R6 (−1522 to −1312bp). Hprt was included as a loading control. Data are representative of two independent experiments. (B) qPCR analysis of Slug transcripts in c-Myc-expressing HSPCs after treatment with SCF. HPSCs harboring c-Myc shRNA and control shRNA were left without treatment as a control or treated with SCF (100 ng/ml) for 12 hrs before qPCR analysis. Data are representative of two independent experiments. **p
    Figure Legend Snippet: Key Role of c-Myc and FoxM1 in SCF/c-Kit-Slug Feedback Loop (A) Analysis of c-Myc and FoxM1 occupancies at the Slug promoter by ChIP analysis. Primary BM cells were transduced with retroviral particles containing c-Myc and FoxM1, respectively. The genomic DNA was pulled down by anti-Flag and IgG control antibodies, and amplified with primer pairs F1/R1 (−856 to −691bp), F2/R2 (−1389 to −1234bp), F3/R3 (−1772 to −1605bp), F4/R4 (−597 to −410bp) F5/R5 (−1203 to −1012bp), F6/R6 (−1522 to −1312bp). Hprt was included as a loading control. Data are representative of two independent experiments. (B) qPCR analysis of Slug transcripts in c-Myc-expressing HSPCs after treatment with SCF. HPSCs harboring c-Myc shRNA and control shRNA were left without treatment as a control or treated with SCF (100 ng/ml) for 12 hrs before qPCR analysis. Data are representative of two independent experiments. **p

    Techniques Used: Chromatin Immunoprecipitation, Transduction, Amplification, Real-time Polymerase Chain Reaction, Expressing, shRNA

    41) Product Images from "Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles-associated insect-specific flaviviruses"

    Article Title: Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles-associated insect-specific flaviviruses

    Journal: Virus Evolution

    doi: 10.1093/ve/vew035

    In vivo detection of the An. sinensis flavivirus-derived EVE. (A) EVE detection in genomic DNA from the Korean strain of An. sinensis . Lane 1, size marker; lane 2, amplified genomic DNA from a pool of 10 An. minimus adult females; lane 3, amplified genomic DNA from a pool of 10 An. minimus adult males; lane 4, amplified genomic DNA from a pool of 10 An. sinensis adult females; lane 5, amplified genomic DNA from a pool of 10 An. sinensis adult males; lane 6, amplified DNA from a pool of 10 An. stephensi females; lane 7, NTC. (B) EVE detection in total RNA from the Korean strain of An. sinensis . Lane 1: size marker; lanes 2 and 3, amplified cDNA from pools of five adult females; lanes 4 and 5, amplified cDNA from pools of five adult males; lanes 6 and 7, amplified cDNA from pools of five L 4 larvae; lane 8, amplified DNA from a pool of ten females; lane 9, amplified cDNA from a pool of five An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of five adult females as lane 2; lane 11, NTC. First row, EVE; second row, RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product.
    Figure Legend Snippet: In vivo detection of the An. sinensis flavivirus-derived EVE. (A) EVE detection in genomic DNA from the Korean strain of An. sinensis . Lane 1, size marker; lane 2, amplified genomic DNA from a pool of 10 An. minimus adult females; lane 3, amplified genomic DNA from a pool of 10 An. minimus adult males; lane 4, amplified genomic DNA from a pool of 10 An. sinensis adult females; lane 5, amplified genomic DNA from a pool of 10 An. sinensis adult males; lane 6, amplified DNA from a pool of 10 An. stephensi females; lane 7, NTC. (B) EVE detection in total RNA from the Korean strain of An. sinensis . Lane 1: size marker; lanes 2 and 3, amplified cDNA from pools of five adult females; lanes 4 and 5, amplified cDNA from pools of five adult males; lanes 6 and 7, amplified cDNA from pools of five L 4 larvae; lane 8, amplified DNA from a pool of ten females; lane 9, amplified cDNA from a pool of five An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of five adult females as lane 2; lane 11, NTC. First row, EVE; second row, RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product.

    Techniques Used: In Vivo, Derivative Assay, Marker, Amplification, Sequencing, Polymerase Chain Reaction

    In vivo detection of the An. minimus flavivirus-derived EVE. (A) EVE detection in genomic DNA. Lane 1: size marker; lane 2: amplified genomic DNA from a pool of 10 An. minimus adult females; lane 3: amplified genomic DNA from a pool of 10 An. minimus adult males; lane 4: amplified genomic DNA from a pool of 10 An. sinensis adult females; lane 5: amplified genomic DNA from a pool of 10 An. sinensis adult males; lane 6: amplified DNA from a pool of 10 An. stephensi females; lane 7: no template control (NTC). (B) EVE detection in total RNA. Lane 1: size marker; lanes 2 and 3: amplified cDNA from pools of five adult females; lanes 4 and 5: amplified cDNA from pools of five adult males; lanes 6 and 7: amplified cDNA from pools of five L 4 larvae; lane 8: amplified DNA from a pool of 10 females; lane 9: amplified cDNA from a pool of 5 An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of five adult females as lane 2; lane 11: NTC. First row: EVE; second row: RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product.
    Figure Legend Snippet: In vivo detection of the An. minimus flavivirus-derived EVE. (A) EVE detection in genomic DNA. Lane 1: size marker; lane 2: amplified genomic DNA from a pool of 10 An. minimus adult females; lane 3: amplified genomic DNA from a pool of 10 An. minimus adult males; lane 4: amplified genomic DNA from a pool of 10 An. sinensis adult females; lane 5: amplified genomic DNA from a pool of 10 An. sinensis adult males; lane 6: amplified DNA from a pool of 10 An. stephensi females; lane 7: no template control (NTC). (B) EVE detection in total RNA. Lane 1: size marker; lanes 2 and 3: amplified cDNA from pools of five adult females; lanes 4 and 5: amplified cDNA from pools of five adult males; lanes 6 and 7: amplified cDNA from pools of five L 4 larvae; lane 8: amplified DNA from a pool of 10 females; lane 9: amplified cDNA from a pool of 5 An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of five adult females as lane 2; lane 11: NTC. First row: EVE; second row: RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product.

    Techniques Used: In Vivo, Derivative Assay, Marker, Amplification, Sequencing, Polymerase Chain Reaction

    42) Product Images from "The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN"

    Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.024448

    Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ- 32 P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC 50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).
    Figure Legend Snippet: Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ- 32 P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC 50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

    Techniques Used: Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

    43) Product Images from "Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway"

    Article Title: Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174017

    HNF1α inhibits HBV gene expression and replication through activating the NF-κB signaling. Huh7 cells cultured in 6-well plate were co-transfected with the indicated amount of the plasmids. (A) The replication intermediates in viral core particles were examined with Southern blot and Viral RNAs with Northern blot. RC, relaxed circular DNA. 18S/28S RNAs served as the RNA loading control. HBV replication intermediates were quantified using densitometry scanning. Transfection efficiency was normalized by using co-transfected pGL3-luciferase plasmid and control measurements were taken as 100%. (B) The culture supernatants were collected to detect HBsAg, HBeAg and HBV DNA. Means and SEMs of data from three independent experiments are plotted. * P
    Figure Legend Snippet: HNF1α inhibits HBV gene expression and replication through activating the NF-κB signaling. Huh7 cells cultured in 6-well plate were co-transfected with the indicated amount of the plasmids. (A) The replication intermediates in viral core particles were examined with Southern blot and Viral RNAs with Northern blot. RC, relaxed circular DNA. 18S/28S RNAs served as the RNA loading control. HBV replication intermediates were quantified using densitometry scanning. Transfection efficiency was normalized by using co-transfected pGL3-luciferase plasmid and control measurements were taken as 100%. (B) The culture supernatants were collected to detect HBsAg, HBeAg and HBV DNA. Means and SEMs of data from three independent experiments are plotted. * P

    Techniques Used: Expressing, Cell Culture, Transfection, Southern Blot, Northern Blot, Luciferase, Plasmid Preparation

    HNF1α inhibits HBV gene expression and replication in Huh7 cells. (A) HNF1α overexpression inhibited HBV antigen and DNA productions. Huh7 cells cultured in 24-well plate were co-transfected with the indicated plasmids. 48 hours post-transfection, the extracellular levels of HBsAg, HBeAg and HBV DNA and the intracellular levels of HBsAg and HBeAg were measured. (B) Knockdown of endogenous HNF1α expression enhanced HBV antigen and DNA productions. Huh7 cells cultured in 6-well plate were transduced by the lentivirus expressing sh-HNF1α (+ and ++ stand for 0.5 ml and 1 ml lentivirus supernatant per well, respectively) or sh-EGFP. 16 hours post-transduction, cells were transfected with pHBV1.3 (1 μg). The extracellular levels of HBsAg, HBeAg and HBV DNA were determined. Means and SEMs of data from at least three independent tests were plotted. * P
    Figure Legend Snippet: HNF1α inhibits HBV gene expression and replication in Huh7 cells. (A) HNF1α overexpression inhibited HBV antigen and DNA productions. Huh7 cells cultured in 24-well plate were co-transfected with the indicated plasmids. 48 hours post-transfection, the extracellular levels of HBsAg, HBeAg and HBV DNA and the intracellular levels of HBsAg and HBeAg were measured. (B) Knockdown of endogenous HNF1α expression enhanced HBV antigen and DNA productions. Huh7 cells cultured in 6-well plate were transduced by the lentivirus expressing sh-HNF1α (+ and ++ stand for 0.5 ml and 1 ml lentivirus supernatant per well, respectively) or sh-EGFP. 16 hours post-transduction, cells were transfected with pHBV1.3 (1 μg). The extracellular levels of HBsAg, HBeAg and HBV DNA were determined. Means and SEMs of data from at least three independent tests were plotted. * P

    Techniques Used: Expressing, Over Expression, Cell Culture, Transfection, Transduction

    44) Product Images from "Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA"

    Article Title: Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203953

    Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.
    Figure Legend Snippet: Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Techniques Used: Generated, Sequencing

    45) Product Images from "Dimerized translationally controlled tumor protein increases interleukin-8 expression through MAPK and NF-κB pathways in a human bronchial epithelial cell line"

    Article Title: Dimerized translationally controlled tumor protein increases interleukin-8 expression through MAPK and NF-κB pathways in a human bronchial epithelial cell line

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-018-0214-6

    The role of NF-κB and AP-1 in dTCTP-induced IL-8 production. Electrophoretic mobility shift assays to measure the DNA-binding activities of NF-κB ( a ) and AP-1 ( b ) binding to their recognition sites were performed. Nuclear extracts were prepared from the BEAS-2B cells stimulated with 10 μg/ml of dTCTP for various time periods (0, 30 and 60 min). EMSA was performed using nuclear extract binding buffer (100 mm Tris, 500 mm KCl and 10 mm DTT; pH 7.5), and biotinylated probes were incubated at room temperature for 20 min. The protein–DNA complexes were electrophoresed on 6% polyacrylamide gels at 4 °C in ×0.5 TBE buffer and transferred to a nylon membrane. The UV cross-linked membrane was treated with streptavidin–horseradish peroxidase conjugate and then detected with CCD camera imaging device using the LightShift Chemiluminescent EMSA Kit. Unlabelled oligonucleotides were included to examine binding specificity. c Schematic presentation of IL-8 promoter luciferase reporter system and two mutant IL-8 promoters with an altered NF-κB or AP-1 binding site (left panel). Luciferase activities of BEAS-2B cells carrying IL-8 promoter reporter plasmids were measured using dual luciferase assay system. BEAS-2B cells were transiently transfected by Lipofectamine™ 2000 reagent with 1 μg of each IL-8 luciferase plasmids and 0.1 μg of pRL-TK. At 24 h after transfection, the cells were stimulated for 24 h with dTCTP (10 μg/ml). Values are expressed as fold changes (mean ± SEM, n = 3). **p
    Figure Legend Snippet: The role of NF-κB and AP-1 in dTCTP-induced IL-8 production. Electrophoretic mobility shift assays to measure the DNA-binding activities of NF-κB ( a ) and AP-1 ( b ) binding to their recognition sites were performed. Nuclear extracts were prepared from the BEAS-2B cells stimulated with 10 μg/ml of dTCTP for various time periods (0, 30 and 60 min). EMSA was performed using nuclear extract binding buffer (100 mm Tris, 500 mm KCl and 10 mm DTT; pH 7.5), and biotinylated probes were incubated at room temperature for 20 min. The protein–DNA complexes were electrophoresed on 6% polyacrylamide gels at 4 °C in ×0.5 TBE buffer and transferred to a nylon membrane. The UV cross-linked membrane was treated with streptavidin–horseradish peroxidase conjugate and then detected with CCD camera imaging device using the LightShift Chemiluminescent EMSA Kit. Unlabelled oligonucleotides were included to examine binding specificity. c Schematic presentation of IL-8 promoter luciferase reporter system and two mutant IL-8 promoters with an altered NF-κB or AP-1 binding site (left panel). Luciferase activities of BEAS-2B cells carrying IL-8 promoter reporter plasmids were measured using dual luciferase assay system. BEAS-2B cells were transiently transfected by Lipofectamine™ 2000 reagent with 1 μg of each IL-8 luciferase plasmids and 0.1 μg of pRL-TK. At 24 h after transfection, the cells were stimulated for 24 h with dTCTP (10 μg/ml). Values are expressed as fold changes (mean ± SEM, n = 3). **p

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Imaging, Luciferase, Mutagenesis, Transfection

    46) Product Images from "Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection"

    Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00887-18

    Genetic deletion of B. burgdorferi bbk13 . (A) Schematic representation of the linear plasmid 36 (lp36) genomic locus containing bbk13 and adjacent genes. The bbk13 open reading frame (orange arrow) was replaced with the flaBp-aadA antibiotic resistance cassette (black arrow) in wild-type B. burgdorferi (WT) by allelic exchange. Deletion boundaries are indicated by the black dashed lines. Complementation of the bbk13 mutant (Δ bbk13 ) with a wild-type copy of bbk13 under the control of its putative endogenous promoter (the DNA region indicated by the green dotted-line box) on pBSV2G was performed in trans , resulting in the Δ bbk13 / bbk13 + clone. The bbk13 ) is indicated by a bent black arrow at position 8,903 bp. (B) bbk13 expression is abolished in the Δ bbk13 mutant and restored in the Δ bbk13 / bbk13 + complemented clone. RNA was isolated from WT, Δ bbk13 , and Δ bbk13 / bbk13 + clones, and bbk13 expression was measured by RT-qPCR. Gene expression of recA was used for normalization. The mean ± standard deviation from three biological replicates are shown. Statistical significance was tested using one-way ANOVA and Tukey’s multiple-comparison test (*, P  
    Figure Legend Snippet: Genetic deletion of B. burgdorferi bbk13 . (A) Schematic representation of the linear plasmid 36 (lp36) genomic locus containing bbk13 and adjacent genes. The bbk13 open reading frame (orange arrow) was replaced with the flaBp-aadA antibiotic resistance cassette (black arrow) in wild-type B. burgdorferi (WT) by allelic exchange. Deletion boundaries are indicated by the black dashed lines. Complementation of the bbk13 mutant (Δ bbk13 ) with a wild-type copy of bbk13 under the control of its putative endogenous promoter (the DNA region indicated by the green dotted-line box) on pBSV2G was performed in trans , resulting in the Δ bbk13 / bbk13 + clone. The bbk13 ) is indicated by a bent black arrow at position 8,903 bp. (B) bbk13 expression is abolished in the Δ bbk13 mutant and restored in the Δ bbk13 / bbk13 + complemented clone. RNA was isolated from WT, Δ bbk13 , and Δ bbk13 / bbk13 + clones, and bbk13 expression was measured by RT-qPCR. Gene expression of recA was used for normalization. The mean ± standard deviation from three biological replicates are shown. Statistical significance was tested using one-way ANOVA and Tukey’s multiple-comparison test (*, P  

    Techniques Used: Plasmid Preparation, Mutagenesis, Expressing, Isolation, Clone Assay, Quantitative RT-PCR, Standard Deviation

    47) Product Images from "Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission"

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004350

    Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc) by crossing healthy female (n = 8) mice with infected male mice (n = 5). BLI signal from ex vivo organs of female mice (n = 8) crossed with T . b . gambiense 1135 infected males (n = 5) examined 7 months after crossing. A. BLI of ex vivo organs of a representative female mouse (mouse 229, see S3 Table ). Ov, ovaries; Ut, uterus; B, brain; SC, spinal cord; Sp, spleen; Li, liver; Lu, lungs; K, kidneys; In, intestines; H, heart. The colour scale to the right of the image indicates the colour intensity in ph/sr/cm 2 /s. B. Percentage of positive female mice per organ. C. 1.8% agarose gel of PCR using pMUTec/TBingi nested primers on organs of female mice 229 and 228. Lane 1: spinal cord negative control; lane 2: spleen negative control; lane 3: ovary mouse 229; lane 4: uterus mouse 229 (band 201 bp); lane 5: spinal cord negative control; lane 6 spinal cord mouse 229; lane 7: spleen mouse 229; lane 8: ovary mouse 228; lane 9: uterus mouse 228; lane 10: spinal cord mouse 228; lane 11: genomic DNA T . b . brucei (nested PCR of the first PCR positive control); lane 12: genomic DNA T . b . brucei (nested PCR positive control); lane M: GeneRuler DNA ladder (ThermoScientific).
    Figure Legend Snippet: Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc) by crossing healthy female (n = 8) mice with infected male mice (n = 5). BLI signal from ex vivo organs of female mice (n = 8) crossed with T . b . gambiense 1135 infected males (n = 5) examined 7 months after crossing. A. BLI of ex vivo organs of a representative female mouse (mouse 229, see S3 Table ). Ov, ovaries; Ut, uterus; B, brain; SC, spinal cord; Sp, spleen; Li, liver; Lu, lungs; K, kidneys; In, intestines; H, heart. The colour scale to the right of the image indicates the colour intensity in ph/sr/cm 2 /s. B. Percentage of positive female mice per organ. C. 1.8% agarose gel of PCR using pMUTec/TBingi nested primers on organs of female mice 229 and 228. Lane 1: spinal cord negative control; lane 2: spleen negative control; lane 3: ovary mouse 229; lane 4: uterus mouse 229 (band 201 bp); lane 5: spinal cord negative control; lane 6 spinal cord mouse 229; lane 7: spleen mouse 229; lane 8: ovary mouse 228; lane 9: uterus mouse 228; lane 10: spinal cord mouse 228; lane 11: genomic DNA T . b . brucei (nested PCR of the first PCR positive control); lane 12: genomic DNA T . b . brucei (nested PCR positive control); lane M: GeneRuler DNA ladder (ThermoScientific).

    Techniques Used: Transmission Assay, Mouse Assay, Infection, Ex Vivo, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Nested PCR, Positive Control

    48) Product Images from "NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks *"

    Article Title: NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.456087

    NEIL1 binding to interstrand crosslink DNA. A , NEIL1 protein titration. NEIL1 protein was incubated at increasing concentrations with the positive control 5OHC-containing duplex substrate, and the binding reactions were resolved on a native gel. Open
    Figure Legend Snippet: NEIL1 binding to interstrand crosslink DNA. A , NEIL1 protein titration. NEIL1 protein was incubated at increasing concentrations with the positive control 5OHC-containing duplex substrate, and the binding reactions were resolved on a native gel. Open

    Techniques Used: Binding Assay, Titration, Incubation, Positive Control

    49) Product Images from "Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery"

    Article Title: Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

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

    doi: 10.1073/pnas.1705508114

    T4SS-like inner-membrane complex is required for the release of DNA from NTHI. Time-lapse fluorescence microscopy images of the Δ traCG mutant and the complemented Δ traCG mutant are shown. Time stamps indicate elapsed incubation time. ( A ) No DNA was released from the Δ traCG mutant at any time point. However, a subset of cells (dashed boxes) had taken up the ethidium homodimer-2 DNA stain. (Scale bar, 10 µm.) ( A , Inset ) Magnified image of a Δ traCG mutant cell showed a clear demarcation between red fluorescent DNA and green fluorescent outer membrane. (Scale bar, 2 μm.) ( B ) Complementation of the Δ traCG mutant restored the characteristic DNA flare release phenotype (dashed boxes). (Scale bar, 10 µm.) ( B , Inset ) Magnified image of a complemented Δ traCG mutant cell showed the characteristic red flare release phenotype (blue bracket). (Scale bar, 2 μm.)
    Figure Legend Snippet: T4SS-like inner-membrane complex is required for the release of DNA from NTHI. Time-lapse fluorescence microscopy images of the Δ traCG mutant and the complemented Δ traCG mutant are shown. Time stamps indicate elapsed incubation time. ( A ) No DNA was released from the Δ traCG mutant at any time point. However, a subset of cells (dashed boxes) had taken up the ethidium homodimer-2 DNA stain. (Scale bar, 10 µm.) ( A , Inset ) Magnified image of a Δ traCG mutant cell showed a clear demarcation between red fluorescent DNA and green fluorescent outer membrane. (Scale bar, 2 μm.) ( B ) Complementation of the Δ traCG mutant restored the characteristic DNA flare release phenotype (dashed boxes). (Scale bar, 10 µm.) ( B , Inset ) Magnified image of a complemented Δ traCG mutant cell showed the characteristic red flare release phenotype (blue bracket). (Scale bar, 2 μm.)

    Techniques Used: Fluorescence, Microscopy, Mutagenesis, Incubation, Staining

    Expression of ComE, but not PilA, is required for the release of DNA by NTHI. Time-lapse fluorescence microscopy images of the Δ comE mutant and the complemented Δ comE mutant are shown. Time stamps indicate elapsed incubation time. ( A ) No DNA was released from the Δ comE mutant at any time point; however, a subset of cells (dashed boxes) was uniformly red due to dye uptake. (Scale bar, 10 µm.) ( A , Inset ) Magnified image of a uniformly red fluorescent cell. (Scale bar, 2 μm.) ( B ) Complementation of the Δ comE mutant restored the DNA flare release phenotype (dashed boxes). (Scale bar, 10 µm.) ( B , Inset ) Magnified image of a complemented Δ comE mutant cell with the characteristic red flare (blue bracket). (Scale bar, 2 μm.) ( C ) DNA was released from Δ pilA cells via the characteristic red flare phenotype (dashed boxes), which indicated that deletion of this component of the Tfp machinery did not interfere with the described release mechanism. (Scale bar, 10 µm.) ( C , Inset ) Magnified image of a Δ pilA mutant cell with the characteristic red flare (blue bracket). (Scale bar, 2 μm.)
    Figure Legend Snippet: Expression of ComE, but not PilA, is required for the release of DNA by NTHI. Time-lapse fluorescence microscopy images of the Δ comE mutant and the complemented Δ comE mutant are shown. Time stamps indicate elapsed incubation time. ( A ) No DNA was released from the Δ comE mutant at any time point; however, a subset of cells (dashed boxes) was uniformly red due to dye uptake. (Scale bar, 10 µm.) ( A , Inset ) Magnified image of a uniformly red fluorescent cell. (Scale bar, 2 μm.) ( B ) Complementation of the Δ comE mutant restored the DNA flare release phenotype (dashed boxes). (Scale bar, 10 µm.) ( B , Inset ) Magnified image of a complemented Δ comE mutant cell with the characteristic red flare (blue bracket). (Scale bar, 2 μm.) ( C ) DNA was released from Δ pilA cells via the characteristic red flare phenotype (dashed boxes), which indicated that deletion of this component of the Tfp machinery did not interfere with the described release mechanism. (Scale bar, 10 µm.) ( C , Inset ) Magnified image of a Δ pilA mutant cell with the characteristic red flare (blue bracket). (Scale bar, 2 μm.)

    Techniques Used: Expressing, Fluorescence, Microscopy, Mutagenesis, Incubation

    Subpopulation of NTHI released DNA from a single subpolar location along one long axis of the cell. ( A ) Time-lapse fluorescence video microscopy demonstrated DNA release from NTHI via a mechanism that did not involve development of large round cells or explosive cell lysis. Representative images show NTHI (green) cultured in medium that contained the membrane-impermeable dsDNA stain ethidium homodimer-2 (red), and thus a fluorescent red flare indicated DNA release from the bacterial cell (dashed boxes). Representative still frame was taken after 50 min of incubation. (Scale bar, 10 µm.) ( A , Inset ) Image of a cell with a red flare that indicated DNA release (blue bracket). (Scale bar, 2 μm.) ( B ) To validate that DNA was released from the bacterial cell, intracellular DNA was first stained with the cell-permeable stain Syto 9, which fluoresces green when bound to DNA, and the bacterial outer membrane was stained with FM4-64 (a red fluorescent membrane stain). Fluorescence time-lapse microscopy images confirmed that DNA was released from a subpolar location of a subset of cells. The presence of eDNA (now green) adjacent to a bacterial cell (now red) supported the proposed release mechanism (dashed boxes). (Scale bar, 10 μm.) ( B , Inset ) Magnified image of bacteria with a green flare that indicated DNA release (blue bracket). (Scale bar, 2 μm.)
    Figure Legend Snippet: Subpopulation of NTHI released DNA from a single subpolar location along one long axis of the cell. ( A ) Time-lapse fluorescence video microscopy demonstrated DNA release from NTHI via a mechanism that did not involve development of large round cells or explosive cell lysis. Representative images show NTHI (green) cultured in medium that contained the membrane-impermeable dsDNA stain ethidium homodimer-2 (red), and thus a fluorescent red flare indicated DNA release from the bacterial cell (dashed boxes). Representative still frame was taken after 50 min of incubation. (Scale bar, 10 µm.) ( A , Inset ) Image of a cell with a red flare that indicated DNA release (blue bracket). (Scale bar, 2 μm.) ( B ) To validate that DNA was released from the bacterial cell, intracellular DNA was first stained with the cell-permeable stain Syto 9, which fluoresces green when bound to DNA, and the bacterial outer membrane was stained with FM4-64 (a red fluorescent membrane stain). Fluorescence time-lapse microscopy images confirmed that DNA was released from a subpolar location of a subset of cells. The presence of eDNA (now green) adjacent to a bacterial cell (now red) supported the proposed release mechanism (dashed boxes). (Scale bar, 10 μm.) ( B , Inset ) Magnified image of bacteria with a green flare that indicated DNA release (blue bracket). (Scale bar, 2 μm.)

    Techniques Used: Fluorescence, Microscopy, Lysis, Cell Culture, Staining, Incubation, Time-lapse Microscopy

    Representative STEM images of parent, Δ comE , and Δ traCG cells labeled for DNA. Ultrathin sections of bacterial cells were immunolabeled with monoclonal antibody against dsDNA and revealed with goat anti-mouse IgG conjugated to 6-nm gold. Sections were imaged by scanning transmission electron microscopy ( A , C , E , and G ), and secondary electron detection ( B , D , F , and H ) as an additional method to validate the presence and precise location of gold labeling. ( A – D ) A subset of NTHI parent cells (red boxes) positively labeled for DNA within the cytoplasm, periplasm, and extracellular environment as seen by 6-nm gold particles. ( E and F ) A subset of Δ comE mutant cells (red boxes) positively labeled for DNA within the cytoplasm and in the periplasm, but no extracellular DNA was detected. ( G and H ) DNA was detected within the cytoplasm of the Δ traCG mutant cells (red boxes) but there was no DNA labeled within either the periplasm or extracellular space. (Scale bars, 500 nm.)
    Figure Legend Snippet: Representative STEM images of parent, Δ comE , and Δ traCG cells labeled for DNA. Ultrathin sections of bacterial cells were immunolabeled with monoclonal antibody against dsDNA and revealed with goat anti-mouse IgG conjugated to 6-nm gold. Sections were imaged by scanning transmission electron microscopy ( A , C , E , and G ), and secondary electron detection ( B , D , F , and H ) as an additional method to validate the presence and precise location of gold labeling. ( A – D ) A subset of NTHI parent cells (red boxes) positively labeled for DNA within the cytoplasm, periplasm, and extracellular environment as seen by 6-nm gold particles. ( E and F ) A subset of Δ comE mutant cells (red boxes) positively labeled for DNA within the cytoplasm and in the periplasm, but no extracellular DNA was detected. ( G and H ) DNA was detected within the cytoplasm of the Δ traCG mutant cells (red boxes) but there was no DNA labeled within either the periplasm or extracellular space. (Scale bars, 500 nm.)

    Techniques Used: Labeling, Immunolabeling, Transmission Assay, Electron Microscopy, Mutagenesis

    Imaging release of DNA and DNABII proteins via the ComE secretin of NTHI. ( A ) To confirm the conserved localization of DNA and DNABII release, immunoSTEM of NTHI cells imaged 3 h after inoculation onto the surface of a formvar-coated grid demonstrated the presence of both eDNA (15-nm gold spheres; arrows) and DNABII proteins (6-nm gold spheres) in close proximity to the bacterial cell and at a location that corresponded to the DNA flare seen in fluorescence microscopy. (Scale bar, 250 nm.) ( B ) E. coli that expressed NTHI ComE was immunolabeled to determine the localization of the ComE pore. ComE (green) was localized to a single subpolar location (red), similar to that of DNA and DNABII protein release events shown in A and C . (Scale bar, 2 μm.) ( C ) Low-magnification SEM image of an NTHI cell releasing DNA and DNABII proteins from a single subpolar site and the assembly of these components into a lattice-like matrix. (Scale bar, 500 nm.) These data indicated that the release of DNA and DNABII proteins occurred at a similar location as does expression of the ComE pore.
    Figure Legend Snippet: Imaging release of DNA and DNABII proteins via the ComE secretin of NTHI. ( A ) To confirm the conserved localization of DNA and DNABII release, immunoSTEM of NTHI cells imaged 3 h after inoculation onto the surface of a formvar-coated grid demonstrated the presence of both eDNA (15-nm gold spheres; arrows) and DNABII proteins (6-nm gold spheres) in close proximity to the bacterial cell and at a location that corresponded to the DNA flare seen in fluorescence microscopy. (Scale bar, 250 nm.) ( B ) E. coli that expressed NTHI ComE was immunolabeled to determine the localization of the ComE pore. ComE (green) was localized to a single subpolar location (red), similar to that of DNA and DNABII protein release events shown in A and C . (Scale bar, 2 μm.) ( C ) Low-magnification SEM image of an NTHI cell releasing DNA and DNABII proteins from a single subpolar site and the assembly of these components into a lattice-like matrix. (Scale bar, 500 nm.) These data indicated that the release of DNA and DNABII proteins occurred at a similar location as does expression of the ComE pore.

    Techniques Used: Imaging, Fluorescence, Microscopy, Immunolabeling, Expressing

    Graphic representation of the proposed mechanism for DNA and DNABII protein release from NTHI. In the parental isolate, DNA and DNABII proteins cross the inner membrane (IM) via the Tra complex, and are released from the cell through the ComE pore. In the Δ comE mutant, DNA and DNABII proteins can cross the IM via the Tra complex but are unable to cross the outer membrane (OM) in the absence of the ComE outer-membrane pore. Therefore, no DNA or DNABII proteins would be released outside of the cell via this mechanism. In the Δ traCG mutant, wherein cells lack the T4SS-like inner-membrane Tra complex, DNA and DNABII proteins would be confined to the cytoplasm and unable to cross the IM.
    Figure Legend Snippet: Graphic representation of the proposed mechanism for DNA and DNABII protein release from NTHI. In the parental isolate, DNA and DNABII proteins cross the inner membrane (IM) via the Tra complex, and are released from the cell through the ComE pore. In the Δ comE mutant, DNA and DNABII proteins can cross the IM via the Tra complex but are unable to cross the outer membrane (OM) in the absence of the ComE outer-membrane pore. Therefore, no DNA or DNABII proteins would be released outside of the cell via this mechanism. In the Δ traCG mutant, wherein cells lack the T4SS-like inner-membrane Tra complex, DNA and DNABII proteins would be confined to the cytoplasm and unable to cross the IM.

    Techniques Used: Mutagenesis

    Quantitation of DNA release events by NTHI. In rich medium (sBHI), we observed an average of 17 events per 1,000 parent cells after 2 h (solid red bar). There were no detectable events for either the Δ comE or Δ traCG mutant at this time point. Complementation of the mutations restored the average number of release events similar to that of the parent (compare solid red bar with green, blue, and yellow bars; no statistical differences). Induction of competence by incubation of NTHI in sBHI + cAMP (red striped bar) increased the number of DNA release events. This increase was statistically significant when NTHI was incubated in M-IV medium (red stippled bar). The number of DNA release events was calculated per 1,000 cells. Data are shown as the mean ± SEM from five separate sets of 1,000 cells (5,000 total cells evaluated). Statistical significance was calculated using one-way ANOVA with significance shown at P ≤ 0.001.
    Figure Legend Snippet: Quantitation of DNA release events by NTHI. In rich medium (sBHI), we observed an average of 17 events per 1,000 parent cells after 2 h (solid red bar). There were no detectable events for either the Δ comE or Δ traCG mutant at this time point. Complementation of the mutations restored the average number of release events similar to that of the parent (compare solid red bar with green, blue, and yellow bars; no statistical differences). Induction of competence by incubation of NTHI in sBHI + cAMP (red striped bar) increased the number of DNA release events. This increase was statistically significant when NTHI was incubated in M-IV medium (red stippled bar). The number of DNA release events was calculated per 1,000 cells. Data are shown as the mean ± SEM from five separate sets of 1,000 cells (5,000 total cells evaluated). Statistical significance was calculated using one-way ANOVA with significance shown at P ≤ 0.001.

    Techniques Used: Quantitation Assay, Mutagenesis, Incubation

    eDNA and DNABII proteins were present in biofilms formed by the parental isolate but not the Δ comE or Δ traCG mutants. Biofilms were immunolabeled for DNA and DNABII proteins. ( A – C ) Bacteria within the biofilms were stained with FM1-43 outer-membrane stain (green fluorescence) with both top-down and orthogonal views of representative biofilms shown. ( E – G ) eDNA (white) is visible throughout the biofilm formed by the parental isolate with minimal labeling in biofilms formed by either mutant. ( I – K ) DNABII proteins (red) were visualized in addition to eDNA (white) throughout biofilms formed by the parental isolate. In comparison, minimal to no labeling for DNABII proteins (red) was observed in biofilms formed by either of the mutants. Addition of exogenous NTHI DNA and a DNABII protein (IHF) restored Δ comE biofilm characteristics comparable to the parent (compare D , H , and L with A , E , and I ). (Scale bars, 20 μm.) Note: Mean biofilm height as determined by COMSTAT2 analysis is indicated in the lower left-hand corner of each panel.
    Figure Legend Snippet: eDNA and DNABII proteins were present in biofilms formed by the parental isolate but not the Δ comE or Δ traCG mutants. Biofilms were immunolabeled for DNA and DNABII proteins. ( A – C ) Bacteria within the biofilms were stained with FM1-43 outer-membrane stain (green fluorescence) with both top-down and orthogonal views of representative biofilms shown. ( E – G ) eDNA (white) is visible throughout the biofilm formed by the parental isolate with minimal labeling in biofilms formed by either mutant. ( I – K ) DNABII proteins (red) were visualized in addition to eDNA (white) throughout biofilms formed by the parental isolate. In comparison, minimal to no labeling for DNABII proteins (red) was observed in biofilms formed by either of the mutants. Addition of exogenous NTHI DNA and a DNABII protein (IHF) restored Δ comE biofilm characteristics comparable to the parent (compare D , H , and L with A , E , and I ). (Scale bars, 20 μm.) Note: Mean biofilm height as determined by COMSTAT2 analysis is indicated in the lower left-hand corner of each panel.

    Techniques Used: Immunolabeling, Staining, Fluorescence, Labeling, Mutagenesis, Immunohistofluorescence

    50) Product Images from "Cell cycle‐regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution"

    Article Title: Cell cycle‐regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution

    Journal: The EMBO Journal

    doi: 10.15252/embj.201695135

    RNF8 is required for binding of tankyrase 1 to telomeres and for timely resolution of telomere cohesion A–C Tankyrase 1 association with telomeres is reduced in RNF8‐depleted cells. (A) Immunoblot analysis of HeLaI.2.11 cell lines stably expressing GFP or RNF8 shRNA. (B) Telomeric DNA ChIP analysis of GFP or RNF8 shRNA HeLaI.2.11 cell lines using the indicated antibodies. (C) Quantification of the signal intensity of telomeric DNA immunoprecipitated by anti‐TNKS1 antibody. Average of three independent experiments ± SD. * P ≤ 0.05; Student's unpaired t ‐test. D, E Resolution of telomere cohesion depends on RNF8. (D) Mitotic cells were isolated from HeLaI.2.11 cell lines stably expressing GFP or RNF8 shRNA by mitotic shake‐off and subjected to FISH analysis with a 16ptelo probe. Scale bar, 5 μm. (E) Graphical representation of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments ( n = 60 cells each) ± SEM. ** P ≤ 0.01; Student's unpaired t ‐test. F, G RNF8 depletion leads to sister telomere fusions. (F) Telomere FISH analysis with a (CCCTAA) 3 repeat probe (red) of metaphase spreads from GFP or RNF8 shRNA HTC75 cell lines. DNA was stained with DAPI (blue). White arrowheads indicate sister telomere fusions. Scale bar, 5 μm. (G) Graphical representation of the frequency of sister telomere fusions per chromosome end. Average of two independent experiments ( n = 756–1,012 chromosome ends each) ± SEM. *** P ≤ 0.001; Student's unpaired t ‐test. H DNA ligase IV depletion rescues sister telomere fusions in RNF8 shRNA cells following infection with GFP or Lig4 (47 or 45) shRNA lentivirus. Graphical representation of the frequency of sister telomere fusions per chromosome end. Average of two independent experiments ( n = 702–1,072 chromosome ends each) ± SEM. * P ≤ 0.05; Student's unpaired t ‐test. I Inhibition of ATM reduces K63‐Ub chains on tankyrase 1. HeLaI.2.11 cells were cotransfected with FlagTNKS1 and HA‐K63‐Ub, treated with or without the ATM inhibitor (ATMi) KU‐55933 for 4 h prior to harvest, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies. J, K Inhibition of ATM reduces resolution of telomere cohesion. (J) HeLaI.2.11 cells were isolated by mitotic shake‐off following 4‐h treatment with or without ATMi and subjected to FISH analysis with a 16ptelo probe. Scale bar, 5 μm. (K) Graphical representation of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments ( n = 60 cells each) ± SEM. ** P ≤ 0.01; Student's unpaired t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: RNF8 is required for binding of tankyrase 1 to telomeres and for timely resolution of telomere cohesion A–C Tankyrase 1 association with telomeres is reduced in RNF8‐depleted cells. (A) Immunoblot analysis of HeLaI.2.11 cell lines stably expressing GFP or RNF8 shRNA. (B) Telomeric DNA ChIP analysis of GFP or RNF8 shRNA HeLaI.2.11 cell lines using the indicated antibodies. (C) Quantification of the signal intensity of telomeric DNA immunoprecipitated by anti‐TNKS1 antibody. Average of three independent experiments ± SD. * P ≤ 0.05; Student's unpaired t ‐test. D, E Resolution of telomere cohesion depends on RNF8. (D) Mitotic cells were isolated from HeLaI.2.11 cell lines stably expressing GFP or RNF8 shRNA by mitotic shake‐off and subjected to FISH analysis with a 16ptelo probe. Scale bar, 5 μm. (E) Graphical representation of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments ( n = 60 cells each) ± SEM. ** P ≤ 0.01; Student's unpaired t ‐test. F, G RNF8 depletion leads to sister telomere fusions. (F) Telomere FISH analysis with a (CCCTAA) 3 repeat probe (red) of metaphase spreads from GFP or RNF8 shRNA HTC75 cell lines. DNA was stained with DAPI (blue). White arrowheads indicate sister telomere fusions. Scale bar, 5 μm. (G) Graphical representation of the frequency of sister telomere fusions per chromosome end. Average of two independent experiments ( n = 756–1,012 chromosome ends each) ± SEM. *** P ≤ 0.001; Student's unpaired t ‐test. H DNA ligase IV depletion rescues sister telomere fusions in RNF8 shRNA cells following infection with GFP or Lig4 (47 or 45) shRNA lentivirus. Graphical representation of the frequency of sister telomere fusions per chromosome end. Average of two independent experiments ( n = 702–1,072 chromosome ends each) ± SEM. * P ≤ 0.05; Student's unpaired t ‐test. I Inhibition of ATM reduces K63‐Ub chains on tankyrase 1. HeLaI.2.11 cells were cotransfected with FlagTNKS1 and HA‐K63‐Ub, treated with or without the ATM inhibitor (ATMi) KU‐55933 for 4 h prior to harvest, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies. J, K Inhibition of ATM reduces resolution of telomere cohesion. (J) HeLaI.2.11 cells were isolated by mitotic shake‐off following 4‐h treatment with or without ATMi and subjected to FISH analysis with a 16ptelo probe. Scale bar, 5 μm. (K) Graphical representation of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments ( n = 60 cells each) ± SEM. ** P ≤ 0.01; Student's unpaired t ‐test. Source data are available online for this figure.

    Techniques Used: Binding Assay, Stable Transfection, Expressing, shRNA, Chromatin Immunoprecipitation, Immunoprecipitation, Isolation, Fluorescence In Situ Hybridization, Staining, Infection, Inhibition

    Model showing cell cycle‐regulated control of resolution of telomere cohesion by K63‐ubiquitination of tankyrase 1 In late S/G2 following DNA replication (and in conjunction with the ATM‐mediated DNA damage response at functional telomeres), RNF8 puts K63‐Ub chains on tankyrase 1, promoting its association with telomeres and sister telomere resolution. Following nuclear envelope breakdown, ABRO1 (and BRCC36) gains access to K63‐Ub TNKS1 and removes the chains. ABRO1 remains in the nucleus through G1 keeping the K63‐Ub chains off tankyrase 1 and preventing premature resolution of telomere cohesion in S phase. ABRO1 is exported from the nucleus in S phase to permit K63‐ubiquitination of tankyrase 1. C, cytoplasm; N, nucleus.
    Figure Legend Snippet: Model showing cell cycle‐regulated control of resolution of telomere cohesion by K63‐ubiquitination of tankyrase 1 In late S/G2 following DNA replication (and in conjunction with the ATM‐mediated DNA damage response at functional telomeres), RNF8 puts K63‐Ub chains on tankyrase 1, promoting its association with telomeres and sister telomere resolution. Following nuclear envelope breakdown, ABRO1 (and BRCC36) gains access to K63‐Ub TNKS1 and removes the chains. ABRO1 remains in the nucleus through G1 keeping the K63‐Ub chains off tankyrase 1 and preventing premature resolution of telomere cohesion in S phase. ABRO1 is exported from the nucleus in S phase to permit K63‐ubiquitination of tankyrase 1. C, cytoplasm; N, nucleus.

    Techniques Used: Functional Assay

    51) Product Images from "Therapeutic Peptide Amphiphile as a Drug Carrier with ATP-Triggered Release for Synergistic Effect, Improved Therapeutic Index, and Penetration of 3D Cancer Cell Spheroids"

    Article Title: Therapeutic Peptide Amphiphile as a Drug Carrier with ATP-Triggered Release for Synergistic Effect, Improved Therapeutic Index, and Penetration of 3D Cancer Cell Spheroids

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092773

    ( A ) Schematic illustration of the ATP-triggered release of Dox from the ATP-binding aptamer incorporated DNA scaffold. ( B ) The fluorescence spectra of Dox (1 µg/mL) with increasing mass ratios of the DNA scaffold. ( C ) The percentage of Dox fluorescence recovery from the Dox-DNA in the presence of different concentrations of ATP (1, 4, 8, 20 mM). Error bars represent SD ( n = 3). ( D ) the fluorescence spectra of Dox-DNA (1 µg/mL) at the mass ratio of Dox to DNA of 1:10 in the presence of different concentrations of ATP (1, 4, 8, 20 mM).
    Figure Legend Snippet: ( A ) Schematic illustration of the ATP-triggered release of Dox from the ATP-binding aptamer incorporated DNA scaffold. ( B ) The fluorescence spectra of Dox (1 µg/mL) with increasing mass ratios of the DNA scaffold. ( C ) The percentage of Dox fluorescence recovery from the Dox-DNA in the presence of different concentrations of ATP (1, 4, 8, 20 mM). Error bars represent SD ( n = 3). ( D ) the fluorescence spectra of Dox-DNA (1 µg/mL) at the mass ratio of Dox to DNA of 1:10 in the presence of different concentrations of ATP (1, 4, 8, 20 mM).

    Techniques Used: Binding Assay, Fluorescence

    ( A ) Schematic illustration of the mechanism for the formation of the Dox-DNA/PAH6 NC. ( B ) The averaged particle size and zeta potential of the Dox-DNA/PAH6 NC at different mass ratios of Dox-DNA and PAH6. Error bars represent SD ( n = 3). ( C ) The averaged particle size and polydispersity index (PDI) of the Dox-DNA/PAH6 NC at different mass ratios of PAH6 and Dox-DNA. Error bars represent SD ( n = 3). ( D ) AFM images of the Dox-DNA/PAH6 NC at the mass ratios of Dox-DNA to PAH6 of 11:40 and 11:60, respectively. The scale bar indicates 100 nm. ( E ) The Dox release profile of the Dox-DNA/PAH6 NC in the presence of 8 mM ATP. Error bars represent SD ( n = 5).
    Figure Legend Snippet: ( A ) Schematic illustration of the mechanism for the formation of the Dox-DNA/PAH6 NC. ( B ) The averaged particle size and zeta potential of the Dox-DNA/PAH6 NC at different mass ratios of Dox-DNA and PAH6. Error bars represent SD ( n = 3). ( C ) The averaged particle size and polydispersity index (PDI) of the Dox-DNA/PAH6 NC at different mass ratios of PAH6 and Dox-DNA. Error bars represent SD ( n = 3). ( D ) AFM images of the Dox-DNA/PAH6 NC at the mass ratios of Dox-DNA to PAH6 of 11:40 and 11:60, respectively. The scale bar indicates 100 nm. ( E ) The Dox release profile of the Dox-DNA/PAH6 NC in the presence of 8 mM ATP. Error bars represent SD ( n = 5).

    Techniques Used:

    52) Product Images from "Phospholipid homeostasis, membrane tenacity and survival of Mtb in lipid rich conditions is determined by MmpL11 function"

    Article Title: Phospholipid homeostasis, membrane tenacity and survival of Mtb in lipid rich conditions is determined by MmpL11 function

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26710-z

    Mtb MmpL11 is essential for the intracellular growth fitness of Mtb: ( A,B ) Confirmation of deletion of mmpL11 by Southern hybridisation ( B ) according to the strategy described in ( A ). The black bar denotes the probe binding site in the genomic DNA, kpnI (k) and sacI (s) sites are indicated. ( C ) Ex vivo growth of Wt, ΔM11 and Comp in PMA differentiated THP1 macrophages. ( D ) Growth of Wt and ΔM11 in 7H9 media of pH 5.1 and 7.2. ( E ) Growth of Wt and ΔM11 in 7H9 media containing 0.05% Tyloxapol relative to growth in Tween 80 containing media. Values are mean O.D. (A600) ± SD for N = 3 replicates. F) The bacterial numbers at 0, 5, and 7 days of growth in media containing 0.05% Tyloxapol is shown. Values represent the average values ± SD of one of 2–3 replicate experiments.
    Figure Legend Snippet: Mtb MmpL11 is essential for the intracellular growth fitness of Mtb: ( A,B ) Confirmation of deletion of mmpL11 by Southern hybridisation ( B ) according to the strategy described in ( A ). The black bar denotes the probe binding site in the genomic DNA, kpnI (k) and sacI (s) sites are indicated. ( C ) Ex vivo growth of Wt, ΔM11 and Comp in PMA differentiated THP1 macrophages. ( D ) Growth of Wt and ΔM11 in 7H9 media of pH 5.1 and 7.2. ( E ) Growth of Wt and ΔM11 in 7H9 media containing 0.05% Tyloxapol relative to growth in Tween 80 containing media. Values are mean O.D. (A600) ± SD for N = 3 replicates. F) The bacterial numbers at 0, 5, and 7 days of growth in media containing 0.05% Tyloxapol is shown. Values represent the average values ± SD of one of 2–3 replicate experiments.

    Techniques Used: Hybridization, Binding Assay, Ex Vivo

    53) Product Images from "MicroRNA-144 is regulated by CP2 and decreases COX-2 expression and PGE2 production in mouse ovarian granulosa cells"

    Article Title: MicroRNA-144 is regulated by CP2 and decreases COX-2 expression and PGE2 production in mouse ovarian granulosa cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.24

    ChIP and EMSA showed that CP2 could bind to the miR-144 promoter in vivo and in vitro . ( a ) ChIP assay. The DNA fragments interacting with CP2 protein were pulled down by anti-HA antibodies. DNA isolated from immunoprecipitated material was amplified by PCR as the template. Total chromatin was used as the input. Normal mouse IgG and OCT1 were used as NCs. ( b ) EMSAs. The probes were incubated with nuclear extracts of mGCs in the absence or presence of a 50-fold excess of various competitor DNA oligos (mutant or unlabelled probes) or antibodies. The specific DNA–protein complex and DNA–protein–antibody complex bands are indicated by arrows. The sequences of the various probes are shown under the panel
    Figure Legend Snippet: ChIP and EMSA showed that CP2 could bind to the miR-144 promoter in vivo and in vitro . ( a ) ChIP assay. The DNA fragments interacting with CP2 protein were pulled down by anti-HA antibodies. DNA isolated from immunoprecipitated material was amplified by PCR as the template. Total chromatin was used as the input. Normal mouse IgG and OCT1 were used as NCs. ( b ) EMSAs. The probes were incubated with nuclear extracts of mGCs in the absence or presence of a 50-fold excess of various competitor DNA oligos (mutant or unlabelled probes) or antibodies. The specific DNA–protein complex and DNA–protein–antibody complex bands are indicated by arrows. The sequences of the various probes are shown under the panel

    Techniques Used: Chromatin Immunoprecipitation, In Vivo, In Vitro, Isolation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Incubation, Mutagenesis

    54) Product Images from "Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon"

    Article Title: Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2540-7

    Correlation of packed cell volume (PCV) in cattle blood with the presence of Trypanosoma DNA. a “Non-infected” vs “infected” animals. Cattle are considered “infected”, if the Trypanosoma species as a source of a PCR product was confirmed, excluding bodonid and NI100 products. All other animals were grouped as “non-infected”. The boxes indicate the corresponding 95% confidence intervals. b PCVs of individual cattle, in which DNA of the indicated parasites were detected. Dotted lines indicate the 95% confidence intervals for PCV of animals, in which no trypanosomal DNA was detected; the grey line indicates the threshold PCV of 25%. c PCVs of individual cattle, in which DNA of Bodonidae or NI100 were detected. Dotted lines indicate the 95% confidence intervals for PCV of “non-infected” animals; the grey line indicates the threshold PCV of 25%
    Figure Legend Snippet: Correlation of packed cell volume (PCV) in cattle blood with the presence of Trypanosoma DNA. a “Non-infected” vs “infected” animals. Cattle are considered “infected”, if the Trypanosoma species as a source of a PCR product was confirmed, excluding bodonid and NI100 products. All other animals were grouped as “non-infected”. The boxes indicate the corresponding 95% confidence intervals. b PCVs of individual cattle, in which DNA of the indicated parasites were detected. Dotted lines indicate the 95% confidence intervals for PCV of animals, in which no trypanosomal DNA was detected; the grey line indicates the threshold PCV of 25%. c PCVs of individual cattle, in which DNA of Bodonidae or NI100 were detected. Dotted lines indicate the 95% confidence intervals for PCV of “non-infected” animals; the grey line indicates the threshold PCV of 25%

    Techniques Used: Infection, Polymerase Chain Reaction

    ITS1 amplicons of Trypanosoma species. a Amplicon sizes expected for amplification with generic primers ITS1-InF and ITS1-InR were calculated from available database sequences and crosschecked with sequences from screened samples. b PCR was performed with representative samples containing DNA of the indicated origin and generic ( a ) or specific primers (Table 1 ). Lane M: Marker GeneRuler 50 bp Ladder (Thermo Scientific); Lane 1: NI100 (generic primers); Lane 2: Bodonid (generic primers); Lane 3: T. vivax (generic primers); Lane 4: T. grayi (generic primers); Lane 5: T. theileri (generic primers); Lane 6: T. brucei ssp. (generic primers); Lane 7: T. grayi (specific primers); Lane 8: T. congolense (specific primers); Lane 9: T. congolense forest (specific primers); Lane 10: T. congolense forest and T. congolense kilifi (specific primers); Lane C: control without DNA template
    Figure Legend Snippet: ITS1 amplicons of Trypanosoma species. a Amplicon sizes expected for amplification with generic primers ITS1-InF and ITS1-InR were calculated from available database sequences and crosschecked with sequences from screened samples. b PCR was performed with representative samples containing DNA of the indicated origin and generic ( a ) or specific primers (Table 1 ). Lane M: Marker GeneRuler 50 bp Ladder (Thermo Scientific); Lane 1: NI100 (generic primers); Lane 2: Bodonid (generic primers); Lane 3: T. vivax (generic primers); Lane 4: T. grayi (generic primers); Lane 5: T. theileri (generic primers); Lane 6: T. brucei ssp. (generic primers); Lane 7: T. grayi (specific primers); Lane 8: T. congolense (specific primers); Lane 9: T. congolense forest (specific primers); Lane 10: T. congolense forest and T. congolense kilifi (specific primers); Lane C: control without DNA template

    Techniques Used: Amplification, Polymerase Chain Reaction, Marker

    55) Product Images from "Photodynamic Therapy with Pc 4 Induces Apoptosis of Candida albicans"

    Article Title: Photodynamic Therapy with Pc 4 Induces Apoptosis of Candida albicans

    Journal: Photochemistry and photobiology

    doi: 10.1111/j.1751-1097.2011.00938.x

    Pc 4-PDT-induced apoptosis in C. albicans , as characterized by nuclear and DNA fragmentation
    Figure Legend Snippet: Pc 4-PDT-induced apoptosis in C. albicans , as characterized by nuclear and DNA fragmentation

    Techniques Used:

    56) Product Images from "NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord"

    Article Title: NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord

    Journal: eLife

    doi: 10.7554/eLife.14264

    NOVA1/2 regulate Dcc alternative splicing. ( A ) Schematic of a Dcc minigene containing the genomic DNA between exons 16 and 17. The alternative sequence is shaded gray. Three candidate Nova binding sites, which are YCAY (Y=C/U) clusters, are located within exon 16, intron 16, and exon 17, respectively (dashed lines indicate the number of YCAY repeats). ( B ) Alternative splicing of wildtype and mutant minigenes. The two RT-PCR products correspond to Dcc long and Dcc short , respectively. Two additional RT-PCR products are produced by exon 17 mutations (asterisk), from utilizing two downstream cryptic splice sites. Mutations in intron 16 block NOVA1/2 from increasing Dcc long . ( C ) Alternative splicing of Dcc minigenes containing different numbers of mutations in intron 16. With an increasing number of mutations, NOVA1/2 gradually lose their ability to promote Dcc long . DOI: http://dx.doi.org/10.7554/eLife.14264.024
    Figure Legend Snippet: NOVA1/2 regulate Dcc alternative splicing. ( A ) Schematic of a Dcc minigene containing the genomic DNA between exons 16 and 17. The alternative sequence is shaded gray. Three candidate Nova binding sites, which are YCAY (Y=C/U) clusters, are located within exon 16, intron 16, and exon 17, respectively (dashed lines indicate the number of YCAY repeats). ( B ) Alternative splicing of wildtype and mutant minigenes. The two RT-PCR products correspond to Dcc long and Dcc short , respectively. Two additional RT-PCR products are produced by exon 17 mutations (asterisk), from utilizing two downstream cryptic splice sites. Mutations in intron 16 block NOVA1/2 from increasing Dcc long . ( C ) Alternative splicing of Dcc minigenes containing different numbers of mutations in intron 16. With an increasing number of mutations, NOVA1/2 gradually lose their ability to promote Dcc long . DOI: http://dx.doi.org/10.7554/eLife.14264.024

    Techniques Used: Droplet Countercurrent Chromatography, Sequencing, Binding Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Produced, Blocking Assay

    57) Product Images from "Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation"

    Article Title: Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    Journal: Stem Cells International

    doi: 10.1155/2016/2349261

    LCL-to-iPSC reprogramming and characterization. (a) Schematic diagram of LCL-to-iPSC reprogramming. (b) Morphology of a reprogrammed iPSC colony at 5x, 10x, and 40x original magnifications, respectively. (c) Immunocytochemistry analysis of generated iPSCs showing expression of pluripotency markers. (d) The graphs showing gene expression of core pluripotency markers in LCLs and their reprogrammed iPSCs. (e) PCR analysis of genomic DNA confirms no integration or retention of plasmid genome/transgene in the LCL reprogrammed iPSCs at passages 17–20. (f) Image showing immunocytochemistry analysis of the cells of three embryonic germ layers differentiated from reprogrammed iPSCs using monolayer differentiation protocol. (g) Image showing normal karyotype of an iPSC line. Karyotype analyses of each reprogrammed iPSC line were found to be normal. (h) The differential gene expression graph showing significant downregulation of LCL specific genes.
    Figure Legend Snippet: LCL-to-iPSC reprogramming and characterization. (a) Schematic diagram of LCL-to-iPSC reprogramming. (b) Morphology of a reprogrammed iPSC colony at 5x, 10x, and 40x original magnifications, respectively. (c) Immunocytochemistry analysis of generated iPSCs showing expression of pluripotency markers. (d) The graphs showing gene expression of core pluripotency markers in LCLs and their reprogrammed iPSCs. (e) PCR analysis of genomic DNA confirms no integration or retention of plasmid genome/transgene in the LCL reprogrammed iPSCs at passages 17–20. (f) Image showing immunocytochemistry analysis of the cells of three embryonic germ layers differentiated from reprogrammed iPSCs using monolayer differentiation protocol. (g) Image showing normal karyotype of an iPSC line. Karyotype analyses of each reprogrammed iPSC line were found to be normal. (h) The differential gene expression graph showing significant downregulation of LCL specific genes.

    Techniques Used: Immunocytochemistry, Generated, Expressing, Polymerase Chain Reaction, Plasmid Preparation

    58) Product Images from "Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein"

    Article Title: Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt593

    ( A ) A representative molecule of pUC19 equilibrated in 2D conformation on a freshly cleaved mica with 10 mM Mg 2+ . ( B ) The centerline of the DNA molecule is obtained by tracking the brightest point along the DNA contour. Each point on the centerline is separated by 8 nm. ( C ) Plot of as a function of the separation L . The closed circles are the experimental data obtained by averaging at least 30 molecules. The experimental data are fitted using equation 1 , shown by the red line. The fit gives a persistence length of 56 nm.
    Figure Legend Snippet: ( A ) A representative molecule of pUC19 equilibrated in 2D conformation on a freshly cleaved mica with 10 mM Mg 2+ . ( B ) The centerline of the DNA molecule is obtained by tracking the brightest point along the DNA contour. Each point on the centerline is separated by 8 nm. ( C ) Plot of as a function of the separation L . The closed circles are the experimental data obtained by averaging at least 30 molecules. The experimental data are fitted using equation 1 , shown by the red line. The fit gives a persistence length of 56 nm.

    Techniques Used:

    ( A ) Closed circular relaxed pUC19 after treatment with Topo 1. ( B ) Circular DNA incubated with HU in the ratio 1 dimer: 1 bp and imaged after 45 min and ( C ) 2 h. The scale bars denote 500 nm.
    Figure Legend Snippet: ( A ) Closed circular relaxed pUC19 after treatment with Topo 1. ( B ) Circular DNA incubated with HU in the ratio 1 dimer: 1 bp and imaged after 45 min and ( C ) 2 h. The scale bars denote 500 nm.

    Techniques Used: Incubation

    59) Product Images from "The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication"

    Article Title: The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01875-16

    KS-Bcl-2 is not essential for KSHV DNA replication. (A) 293A cells harboring KSHV IND were treated with 10 μM FK-506 to induce the lytic replication cycle. Cells were harvested and total DNA extracted at different times postinduction. KSHV genome amplification relative to that in uninduced cells was quantified by qPCR. The number of cellular genome copies was also determined and used for normalization. (B) 293A cells carrying KSHV IND were treated with 10 μM FK-506. After 96 h of treatment, total DNA was extracted, and KSHV genome copies were quantified as described above. The experiment was done three times in triplicate. Values are displayed as mean values and standard errors of the means (SEM). Differences between WT KSHV IND and the mutants were not statistically significant (ns; P > 0.05) as determined by Student's t test.
    Figure Legend Snippet: KS-Bcl-2 is not essential for KSHV DNA replication. (A) 293A cells harboring KSHV IND were treated with 10 μM FK-506 to induce the lytic replication cycle. Cells were harvested and total DNA extracted at different times postinduction. KSHV genome amplification relative to that in uninduced cells was quantified by qPCR. The number of cellular genome copies was also determined and used for normalization. (B) 293A cells carrying KSHV IND were treated with 10 μM FK-506. After 96 h of treatment, total DNA was extracted, and KSHV genome copies were quantified as described above. The experiment was done three times in triplicate. Values are displayed as mean values and standard errors of the means (SEM). Differences between WT KSHV IND and the mutants were not statistically significant (ns; P > 0.05) as determined by Student's t test.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction

    60) Product Images from "Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants"

    Article Title: Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants

    Journal: BioNanoScience

    doi: 10.1007/s12668-016-0253-6

    Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.
    Figure Legend Snippet: Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Techniques Used: Polymerase Chain Reaction, Positive Control, Molecular Weight, Mutagenesis

    61) Product Images from "Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure"

    Article Title: Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

    Journal: eLife

    doi: 10.7554/eLife.34039

    Rare apoptosis of amastigotes in host cells. Trypomastigotes from the T. cruzi colombiana strain were used to infect monolayers of Vero cells. Infected cells were fixed, permeabilized and the TUNEL assay was used to detect DNA fragmentation in intracellular amastigotes 72 hr post-infection. DAPI staining was used to identify DNA-containing organelles (nuclei and kinetoplast). ( A ) DNase-treated cells were used as TUNEL-positive controls. ( B and C ) Arrows show examples of the 2–3 apoptotic amastigotes detected upon scanning approximately 1 × 10 6 infected host cells. Scale bars, 5 μm. HCN, host cell nuclei. Results are representative of two independent experiments.
    Figure Legend Snippet: Rare apoptosis of amastigotes in host cells. Trypomastigotes from the T. cruzi colombiana strain were used to infect monolayers of Vero cells. Infected cells were fixed, permeabilized and the TUNEL assay was used to detect DNA fragmentation in intracellular amastigotes 72 hr post-infection. DAPI staining was used to identify DNA-containing organelles (nuclei and kinetoplast). ( A ) DNase-treated cells were used as TUNEL-positive controls. ( B and C ) Arrows show examples of the 2–3 apoptotic amastigotes detected upon scanning approximately 1 × 10 6 infected host cells. Scale bars, 5 μm. HCN, host cell nuclei. Results are representative of two independent experiments.

    Techniques Used: Infection, TUNEL Assay, Staining

    62) Product Images from "Trichloroethylene-induced alterations in DNA methylation were enriched in polycomb protein binding sites in effector/memory CD4+ T cells"

    Article Title: Trichloroethylene-induced alterations in DNA methylation were enriched in polycomb protein binding sites in effector/memory CD4+ T cells

    Journal: Environmental epigenetics

    doi: 10.1093/eep/dvx013

    Chromosome-specific mean DNA methylation levels. ( A ) The results from the RRBS analysis described in Fig. 1 were sorted into individual chromosomes, and presented after binning for average methylation of the CpGs. The area of each histogram was normalized to one to make it easier to compare the chromosomes. ( B ) The RRBS results were presented (as total number of CpG sites in the different bins) after excluding the CpG sites that were either 0–5% or 95–100% methylated
    Figure Legend Snippet: Chromosome-specific mean DNA methylation levels. ( A ) The results from the RRBS analysis described in Fig. 1 were sorted into individual chromosomes, and presented after binning for average methylation of the CpGs. The area of each histogram was normalized to one to make it easier to compare the chromosomes. ( B ) The RRBS results were presented (as total number of CpG sites in the different bins) after excluding the CpG sites that were either 0–5% or 95–100% methylated

    Techniques Used: DNA Methylation Assay, Methylation

    Average DNA methylation levels of all CpGs interrogated. RRBS analysis of the effector/memory CD4 + T cells collected after 40 weeks of adult exposure to TCE was conducted. ( A ) Histograms show the average methylation of all 337 770 CpG sites examined in CD4 + T cells from either control or TCE-treated mice after binning for average methylation (e.g. 0–5% methylation or 20–25% methylation). ( B ) The same histograms are shown without inclusion of the CpGs that were either 0–5% or 95–100% methylated
    Figure Legend Snippet: Average DNA methylation levels of all CpGs interrogated. RRBS analysis of the effector/memory CD4 + T cells collected after 40 weeks of adult exposure to TCE was conducted. ( A ) Histograms show the average methylation of all 337 770 CpG sites examined in CD4 + T cells from either control or TCE-treated mice after binning for average methylation (e.g. 0–5% methylation or 20–25% methylation). ( B ) The same histograms are shown without inclusion of the CpGs that were either 0–5% or 95–100% methylated

    Techniques Used: DNA Methylation Assay, Methylation, Mouse Assay

    63) Product Images from "A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2"

    Article Title: A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1385

    Primase vs. primer-dependent DNA polymerase activity of intact PolpTN2, PolPTNΔ 311–923 and Taq polymerase. ( A ) Intact PolpTN2 (40 ng/µl), ( B ) PolpTN2Δ 311–923 (80 ng/µl) or ( C ) Taq DNA polymerase (0,05 u/µl) were incubated at 70°C in Taq buffer + 0,4 mM dNTP and 2 ng/µl M13mp18 DNA, which was or was not hybridized to the complementary oligonucleotide primer M13 forward ( Supplementary Table S1 ). At t = 0, 5, 10 and 15 min, aliquots were withdrawn and quenched into 25 mM EDTA. The amount of ds DNA synthesized was then determined using Sybr® Green I fluorescence as described in Materials and Methods. Circles, continuous line: synthesis with hybridized primer; squares, dashed line: synthesis without primer. Points are the average of two determinations. The standard deviation is indicated by error bars.
    Figure Legend Snippet: Primase vs. primer-dependent DNA polymerase activity of intact PolpTN2, PolPTNΔ 311–923 and Taq polymerase. ( A ) Intact PolpTN2 (40 ng/µl), ( B ) PolpTN2Δ 311–923 (80 ng/µl) or ( C ) Taq DNA polymerase (0,05 u/µl) were incubated at 70°C in Taq buffer + 0,4 mM dNTP and 2 ng/µl M13mp18 DNA, which was or was not hybridized to the complementary oligonucleotide primer M13 forward ( Supplementary Table S1 ). At t = 0, 5, 10 and 15 min, aliquots were withdrawn and quenched into 25 mM EDTA. The amount of ds DNA synthesized was then determined using Sybr® Green I fluorescence as described in Materials and Methods. Circles, continuous line: synthesis with hybridized primer; squares, dashed line: synthesis without primer. Points are the average of two determinations. The standard deviation is indicated by error bars.

    Techniques Used: Activity Assay, Incubation, Synthesized, SYBR Green Assay, Fluorescence, Standard Deviation

    Efficiency of intact PolpTN2 and PolpTN2Δ 311–923 in priming DNA synthesis by T. nautilus PolB and Taq DNA polymerase. T. nautilus PolB (8 ng/µl) or Taq polymerase (0,05 u/µl) were incubated at 70°C in Taq buffer + 0,4 mM dNTP and 2 ng/µl M13mp18 DNA (without annealed primer) with increasing concentrations of either intact PolpTN2 ( A ) or PolpTN2 Δ 311–923 ( B ). DNA synthesis by intact PolpTN2 or PolpTN2 Δ 311–923 alone was included as a control. At t = 0 and 6 min, aliquots were withdrawn, quenched in 25 mM EDTA and assayed for ds DNA using Sybr® Green I fluorescence. Circles, continuous line: PolpTN2 or PolpTN2 Δ 311–923 alone; squares, dashed line: PolpTN2 or PolpTN2 Δ 311–923 plus Taq DNA polymerase; triangles, dotted line: PolpTN2 or PolpTN2 Δ 311–923 plus T. nautilus PolB DNA polymerase. Points are the average of three determinations. The standard deviation is indicated by error bars.
    Figure Legend Snippet: Efficiency of intact PolpTN2 and PolpTN2Δ 311–923 in priming DNA synthesis by T. nautilus PolB and Taq DNA polymerase. T. nautilus PolB (8 ng/µl) or Taq polymerase (0,05 u/µl) were incubated at 70°C in Taq buffer + 0,4 mM dNTP and 2 ng/µl M13mp18 DNA (without annealed primer) with increasing concentrations of either intact PolpTN2 ( A ) or PolpTN2 Δ 311–923 ( B ). DNA synthesis by intact PolpTN2 or PolpTN2 Δ 311–923 alone was included as a control. At t = 0 and 6 min, aliquots were withdrawn, quenched in 25 mM EDTA and assayed for ds DNA using Sybr® Green I fluorescence. Circles, continuous line: PolpTN2 or PolpTN2 Δ 311–923 alone; squares, dashed line: PolpTN2 or PolpTN2 Δ 311–923 plus Taq DNA polymerase; triangles, dotted line: PolpTN2 or PolpTN2 Δ 311–923 plus T. nautilus PolB DNA polymerase. Points are the average of three determinations. The standard deviation is indicated by error bars.

    Techniques Used: DNA Synthesis, Incubation, SYBR Green Assay, Fluorescence, Standard Deviation

    64) Product Images from "Molecular evidence for the occurrence of tomato leaf curl New Delhi virus on chayote (Sechium edule) in southern India"

    Article Title: Molecular evidence for the occurrence of tomato leaf curl New Delhi virus on chayote (Sechium edule) in southern India

    Journal: VirusDisease

    doi: 10.1007/s13337-017-0403-7

    Phylogenetic analysis of ToLCNDV (TN TDK CHOU1) isolate infecting chayote in Tamil Nadu based on complete DNA A nucleotide sequences with other ToLCNDV isolates. The trees were constructed using the NJ algorithm implemented by MEGA 6.0 with SLCCNV as an outgroup. The bootstrap consensus tree values from 1000 replicates are given at the branch nodes. Branches corresponding to partitions reproduced in less than 70% of bootstrap replicates are collapsed
    Figure Legend Snippet: Phylogenetic analysis of ToLCNDV (TN TDK CHOU1) isolate infecting chayote in Tamil Nadu based on complete DNA A nucleotide sequences with other ToLCNDV isolates. The trees were constructed using the NJ algorithm implemented by MEGA 6.0 with SLCCNV as an outgroup. The bootstrap consensus tree values from 1000 replicates are given at the branch nodes. Branches corresponding to partitions reproduced in less than 70% of bootstrap replicates are collapsed

    Techniques Used: Construct

    65) Product Images from "The Sulfolobus solfataricus GINS Complex Stimulates DNA Binding and Processive DNA Unwinding by Minichromosome Maintenance Helicase"

    Article Title: The Sulfolobus solfataricus GINS Complex Stimulates DNA Binding and Processive DNA Unwinding by Minichromosome Maintenance Helicase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00496-15

    Effect of SsoGINS on DNA binding by SsoMCM. (A, C, and D) Effect of SsoGINS on the gel retardation patterns of SsoMCM on various DNAs. SsoMCM was mixed with a 32 P-labeled forked DNA (D4) (see Table S1 in the supplemental material) (A), a 62-nt random
    Figure Legend Snippet: Effect of SsoGINS on DNA binding by SsoMCM. (A, C, and D) Effect of SsoGINS on the gel retardation patterns of SsoMCM on various DNAs. SsoMCM was mixed with a 32 P-labeled forked DNA (D4) (see Table S1 in the supplemental material) (A), a 62-nt random

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling

    Binding of SsoGINS to DNAs of different structures. SsoGINS was mixed with a 32 P-labeled DNA fragment containing a 42-bp dsDNA region with a 5′ tail (D2) or a 3′ tail (D3) (see Table S1 in the supplemental material), a 32 P-labeled partial
    Figure Legend Snippet: Binding of SsoGINS to DNAs of different structures. SsoGINS was mixed with a 32 P-labeled DNA fragment containing a 42-bp dsDNA region with a 5′ tail (D2) or a 3′ tail (D3) (see Table S1 in the supplemental material), a 32 P-labeled partial

    Techniques Used: Binding Assay, Labeling

    Processivity of SsoMCM. (A and B) DNA unwinding by SsoMCM. SsoMCM (0.2 μM) was incubated with a radiolabeled oligonucleotide substrate (D4) (see Table S1 in the supplemental material) in the presence of increasing amounts of SsoGINS. Reaction
    Figure Legend Snippet: Processivity of SsoMCM. (A and B) DNA unwinding by SsoMCM. SsoMCM (0.2 μM) was incubated with a radiolabeled oligonucleotide substrate (D4) (see Table S1 in the supplemental material) in the presence of increasing amounts of SsoGINS. Reaction

    Techniques Used: Incubation

    66) Product Images from "The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16"

    Article Title: The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    Journal: Journal of Virology

    doi: 10.1128/JVI.02296-13

    Mutations in E8 increase HPV16 episome copy numbers in normal human keratinocytes. (A) Southern blot analysis of low-molecular-weight DNA isolated from two different sets of low-passage-number HPV16 wt (wt) HPV16 E8− (E8−) or HPV16 E8
    Figure Legend Snippet: Mutations in E8 increase HPV16 episome copy numbers in normal human keratinocytes. (A) Southern blot analysis of low-molecular-weight DNA isolated from two different sets of low-passage-number HPV16 wt (wt) HPV16 E8− (E8−) or HPV16 E8

    Techniques Used: Southern Blot, Molecular Weight, Isolation

    E8 mutations increase E4 and L1 protein expression. Frozen sections of 12-day NHK, HPV16 wt (wt), HPV16 E8− (E8−), or HPV16 E8 KWK mt (E8 KWK mt) organotypic cultures were stained with antibodies against HPV16 E4 (E4) or L1. HPV DNA was
    Figure Legend Snippet: E8 mutations increase E4 and L1 protein expression. Frozen sections of 12-day NHK, HPV16 wt (wt), HPV16 E8− (E8−), or HPV16 E8 KWK mt (E8 KWK mt) organotypic cultures were stained with antibodies against HPV16 E4 (E4) or L1. HPV DNA was

    Techniques Used: Expressing, Staining

    E8 mutations do not influence differentiation or expression of cellular DNA replication proteins in organotypic cultures. Frozen sections of 12-day NHK, HPV16 wt (wt), HPV16 E8− (E8−), or HPV16 E8 KWK mt (E8 KWK mt) organotypic cultures
    Figure Legend Snippet: E8 mutations do not influence differentiation or expression of cellular DNA replication proteins in organotypic cultures. Frozen sections of 12-day NHK, HPV16 wt (wt), HPV16 E8− (E8−), or HPV16 E8 KWK mt (E8 KWK mt) organotypic cultures

    Techniques Used: Expressing

    67) Product Images from "A semisynthetic organism engineered for the stable expansion of the genetic alphabet"

    Article Title: A semisynthetic organism engineered for the stable expansion of the genetic alphabet

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

    doi: 10.1073/pnas.1616443114

    ( A ) The sgRNA sequences used to enhance retention of the UBP in (red denotes guide RNA nucleotides mismatched with the DNA target; the position of d TPT3 is denoted by Y and shown in green); hEGFP is a nontarget sgRNA. ( B ) Sanger sequencing chromatogram
    Figure Legend Snippet: ( A ) The sgRNA sequences used to enhance retention of the UBP in (red denotes guide RNA nucleotides mismatched with the DNA target; the position of d TPT3 is denoted by Y and shown in green); hEGFP is a nontarget sgRNA. ( B ) Sanger sequencing chromatogram

    Techniques Used: Sequencing

    UBP retention assay and the effects of transporter and UBP optimization. ( A ) Schematic representation of the biotin shift assay used to determine UBP retention. The plasmid DNA to be analyzed is first amplified in a PCR supplemented with the unnatural
    Figure Legend Snippet: UBP retention assay and the effects of transporter and UBP optimization. ( A ) Schematic representation of the biotin shift assay used to determine UBP retention. The plasmid DNA to be analyzed is first amplified in a PCR supplemented with the unnatural

    Techniques Used: Shift Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    68) Product Images from "CLICK: one-step generation of conditional knockout mice"

    Article Title: CLICK: one-step generation of conditional knockout mice

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4713-y

    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b : Figure S11). c : Figure S2). M: 100 bp DNA ladder marker. d : Table S3)
    Figure Legend Snippet: Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b : Figure S11). c : Figure S2). M: 100 bp DNA ladder marker. d : Table S3)

    Techniques Used: Generated, Electroporation, CRISPR, Knock-Out, Marker

    One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d : Figure S13)
    Figure Legend Snippet: One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d : Figure S13)

    Techniques Used: Knock-Out, In Vitro, Mouse Assay, Polymerase Chain Reaction, Electroporation, Sequencing, Expressing, Marker

    69) Product Images from "Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas"

    Article Title: Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas

    Journal: Virology

    doi: 10.1016/j.virol.2018.05.016

    Efficient Cas9 mediated cleavage of HIV-1-derived indicator constructs. (A) Schematic of the pNL-NLuc-HXB indicator virus. The viral nef gene was replaced with the NLuc indicator gene. Arrows indicate the positions of sgRNA target sites. (B) 293T cells were co-transfected with an expression plasmid expressing Sp Cas9 and an sgRNA specific for the indicated region of the HIV-1 genome, or a control (Ctrl) non-targeting sgRNA. The cells were also co-transfected with the pNL-NLuc-HXB proviral vector. At 72 hpt, the cultures were harvested and NLuc levels determined. The culture receiving the non-targeting sgRNA (Ctrl) was set at 1.0 and the other results normalized to that value. Average of three biological replicates with SD indicated. RLU: relative light units. ( C ) In parallel, Western blots were performed to determine the level of Gag and p24 capsid expression in the transfected cells. Flag indicates the Sp Cas9 protein, which bears a FLAG epitope tag, while Actin was used as a loading control. A representative experiment is shown. ( D ) 293T cells were transfected with expression plasmids encoding CD4 and CXCR4 as well as Sp Cas9 and an sgRNA specific for the indicated region of the HIV-1 genome, or a control (Ctrl) non-targeting sgRNA (black bars). Cells lacking CD4 served as a negative control for non-specific DNA carry over from the virus producer cells. In parallel, 293T cells were co-transfected with these same plasmids as well as a vector expressing a synthetic cDNA (synTat) that encodes wild type HIV-1 Tat protein but lacks the sgRNA target sites present in the WT Tat ORF (grey bars). At 72 hpt, the cells were infected with the replication competent NL-NLuc-HXB indicator virus. A further 72h later, the cells were lysed and NLuc levels determined. All data are normalized to the culture expressing the non-targeting sgRNA in the absence of synTat and are given in RLU. Average of three biological replicates with SD indicated.
    Figure Legend Snippet: Efficient Cas9 mediated cleavage of HIV-1-derived indicator constructs. (A) Schematic of the pNL-NLuc-HXB indicator virus. The viral nef gene was replaced with the NLuc indicator gene. Arrows indicate the positions of sgRNA target sites. (B) 293T cells were co-transfected with an expression plasmid expressing Sp Cas9 and an sgRNA specific for the indicated region of the HIV-1 genome, or a control (Ctrl) non-targeting sgRNA. The cells were also co-transfected with the pNL-NLuc-HXB proviral vector. At 72 hpt, the cultures were harvested and NLuc levels determined. The culture receiving the non-targeting sgRNA (Ctrl) was set at 1.0 and the other results normalized to that value. Average of three biological replicates with SD indicated. RLU: relative light units. ( C ) In parallel, Western blots were performed to determine the level of Gag and p24 capsid expression in the transfected cells. Flag indicates the Sp Cas9 protein, which bears a FLAG epitope tag, while Actin was used as a loading control. A representative experiment is shown. ( D ) 293T cells were transfected with expression plasmids encoding CD4 and CXCR4 as well as Sp Cas9 and an sgRNA specific for the indicated region of the HIV-1 genome, or a control (Ctrl) non-targeting sgRNA (black bars). Cells lacking CD4 served as a negative control for non-specific DNA carry over from the virus producer cells. In parallel, 293T cells were co-transfected with these same plasmids as well as a vector expressing a synthetic cDNA (synTat) that encodes wild type HIV-1 Tat protein but lacks the sgRNA target sites present in the WT Tat ORF (grey bars). At 72 hpt, the cells were infected with the replication competent NL-NLuc-HXB indicator virus. A further 72h later, the cells were lysed and NLuc levels determined. All data are normalized to the culture expressing the non-targeting sgRNA in the absence of synTat and are given in RLU. Average of three biological replicates with SD indicated.

    Techniques Used: Derivative Assay, Construct, Transfection, Expressing, Plasmid Preparation, Western Blot, FLAG-tag, Negative Control, Infection

    Characterization of mutations introduced into TAR by Sp Cas9 cleavage ( A) Sequencing of TAR DNA from cells expressing Sp Cas9 and the TAR1 sgRNA 72 h after infection with the NL-NLuc-HXB indicator virus. At the top, the wild type HIV-1 sequence is shown, with the sgRNA target sequence underlined and the PAM indicated in red. The predicted TAR DNA cleavage site is indicated by an arrow. Missense mutations are indicated in bold and deletions by dashes. ( B ) Similar to panel A except showing the sequence of TAR DNA recovered from HIV-1 infected cells expressing Sp Cas9 and sgRNA TAR2. ( C ) Similar to panel A except showing the TAR DNA sequences recovered from HIV-1 infected cells expressing Sp Cas9 and both sgRNA TAR1 and sgRNA TAR2.
    Figure Legend Snippet: Characterization of mutations introduced into TAR by Sp Cas9 cleavage ( A) Sequencing of TAR DNA from cells expressing Sp Cas9 and the TAR1 sgRNA 72 h after infection with the NL-NLuc-HXB indicator virus. At the top, the wild type HIV-1 sequence is shown, with the sgRNA target sequence underlined and the PAM indicated in red. The predicted TAR DNA cleavage site is indicated by an arrow. Missense mutations are indicated in bold and deletions by dashes. ( B ) Similar to panel A except showing the sequence of TAR DNA recovered from HIV-1 infected cells expressing Sp Cas9 and sgRNA TAR2. ( C ) Similar to panel A except showing the TAR DNA sequences recovered from HIV-1 infected cells expressing Sp Cas9 and both sgRNA TAR1 and sgRNA TAR2.

    Techniques Used: Sequencing, Expressing, Infection

    70) Product Images from "Downregulation of peroxiredoxin-3 by hydrophobic bile acid induces mitochondrial dysfunction and cellular senescence in human trophoblasts"

    Article Title: Downregulation of peroxiredoxin-3 by hydrophobic bile acid induces mitochondrial dysfunction and cellular senescence in human trophoblasts

    Journal: Scientific Reports

    doi: 10.1038/srep38946

    Treatment with bile acids resulted in mitochondrial dysfunction and trophoblast impairment. ( A – D ) HTR8 cells were treated with 50 μM or 100 μM cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) for 24 hours. ( A ) Cell viability, ( B ) ROS production, ( C ) mitochondrial membrane potential (ΔΨm) and ( D ) adenosine triphosphate (ATP) content were assayed using corresponding kits. The ATP level was shown as nmol/mg protein and the others were expressed as arbitrary units. ( E , G ) qRT-PCR assay was applied to detect mitochondrial gene transcripts levels ( MT-CO1, MT-ND1 and MT-ND6 were taken as the representative of mitochondrial genes transcripts) in HTR8 cells treated with 100 μM CA, DCA, CDCA ( E ) and human placental tissues from normal pregnancies or intrahepatic cholestasis of pregnancy patients ( G ). The messenger RNA levels were calculated by normalisation to an internal control 18s rRNA. ( F , H ) Mitochondrial DNA copy number was quantified by quantitative polymerase chain reaction assay with normalisation to genomic DNA level in 100 μM CA, DCA and CDCA treated HTR8 cells ( F ) or human placentas ( H ). All experiments were performed in triplicate. Data were shown as mean ± standard error of mean. Statistical significance was determined by one-way analysis of variance followed by Dunnett’s post-hoc test, two-tailed Student’s t test or Mann-Whitney test. (* p
    Figure Legend Snippet: Treatment with bile acids resulted in mitochondrial dysfunction and trophoblast impairment. ( A – D ) HTR8 cells were treated with 50 μM or 100 μM cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) for 24 hours. ( A ) Cell viability, ( B ) ROS production, ( C ) mitochondrial membrane potential (ΔΨm) and ( D ) adenosine triphosphate (ATP) content were assayed using corresponding kits. The ATP level was shown as nmol/mg protein and the others were expressed as arbitrary units. ( E , G ) qRT-PCR assay was applied to detect mitochondrial gene transcripts levels ( MT-CO1, MT-ND1 and MT-ND6 were taken as the representative of mitochondrial genes transcripts) in HTR8 cells treated with 100 μM CA, DCA, CDCA ( E ) and human placental tissues from normal pregnancies or intrahepatic cholestasis of pregnancy patients ( G ). The messenger RNA levels were calculated by normalisation to an internal control 18s rRNA. ( F , H ) Mitochondrial DNA copy number was quantified by quantitative polymerase chain reaction assay with normalisation to genomic DNA level in 100 μM CA, DCA and CDCA treated HTR8 cells ( F ) or human placentas ( H ). All experiments were performed in triplicate. Data were shown as mean ± standard error of mean. Statistical significance was determined by one-way analysis of variance followed by Dunnett’s post-hoc test, two-tailed Student’s t test or Mann-Whitney test. (* p

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

    Peroxiredoxin-3 (PRDX3) was downregulated in trophoblastic cells by hydrophobic bile acid instead of promoter hypermethylation. ( A ) DNA methylation analysis for detecting PRDX3 promoter methylation level in placentas from normal pregnancies and intrahepatic cholestasis of pregnancy patients. The percentages of methylated DNA were very low in both groups. JAR ( B ) and HTR8 ( C ) cells were treated with 5 μM 5-aza-2′deoxycytidine (AZA), a specific inhibitor of DNA methylation for 72 hours. After harvest, the messenger RNA (mRNA) level of PRDX3 was determined by quantitative reverse transcription - polymerase chain reaction (qRT-PCR) assay. ( D , E ) JAR and HTR8 cells were treated with vehicle (Con), or with μM cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) for 24 hours. The mRNA level of PRDX3 in JAR ( D ) and HTR8 ( E ) was determined by qRT-PCR assay with normalisation to 18s rRNA level. ( F , G ) JAR and HTR8 cells treated with 0, 50, 100 or 200 μM DCA for 24 hours were subjected to qRT-PCR assays for detecting PRDX3 mRNA expression. ( H ) JAR and HTR8 cells were treated with 0, 50, 100 or 200 μM DCA for 24 hours, respectively. After harvest, the protein level of PRDX3 in JAR and HTR8 cell was assayed by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. Full-length blots were presented in Supplementary Fig. S8 . ( I , J ) Villous explants treated with 0, 10, 50 or 100 μM CA/DCA/CDCA for 24 hours were subjected to qRT-PCR assay for detecting PRDX3 mRNA expression ( I ) and Western blot assay for detecting PRDX3 protein level ( J ). Data were shown as mean ± standard error of mean. Statistical significance was determined by Mann-Whitney test, Student’s t test or one-way analysis of variance followed by Dunnett’s post-hoc test. (ns: no significance; * p
    Figure Legend Snippet: Peroxiredoxin-3 (PRDX3) was downregulated in trophoblastic cells by hydrophobic bile acid instead of promoter hypermethylation. ( A ) DNA methylation analysis for detecting PRDX3 promoter methylation level in placentas from normal pregnancies and intrahepatic cholestasis of pregnancy patients. The percentages of methylated DNA were very low in both groups. JAR ( B ) and HTR8 ( C ) cells were treated with 5 μM 5-aza-2′deoxycytidine (AZA), a specific inhibitor of DNA methylation for 72 hours. After harvest, the messenger RNA (mRNA) level of PRDX3 was determined by quantitative reverse transcription - polymerase chain reaction (qRT-PCR) assay. ( D , E ) JAR and HTR8 cells were treated with vehicle (Con), or with μM cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) for 24 hours. The mRNA level of PRDX3 in JAR ( D ) and HTR8 ( E ) was determined by qRT-PCR assay with normalisation to 18s rRNA level. ( F , G ) JAR and HTR8 cells treated with 0, 50, 100 or 200 μM DCA for 24 hours were subjected to qRT-PCR assays for detecting PRDX3 mRNA expression. ( H ) JAR and HTR8 cells were treated with 0, 50, 100 or 200 μM DCA for 24 hours, respectively. After harvest, the protein level of PRDX3 in JAR and HTR8 cell was assayed by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. Full-length blots were presented in Supplementary Fig. S8 . ( I , J ) Villous explants treated with 0, 10, 50 or 100 μM CA/DCA/CDCA for 24 hours were subjected to qRT-PCR assay for detecting PRDX3 mRNA expression ( I ) and Western blot assay for detecting PRDX3 protein level ( J ). Data were shown as mean ± standard error of mean. Statistical significance was determined by Mann-Whitney test, Student’s t test or one-way analysis of variance followed by Dunnett’s post-hoc test. (ns: no significance; * p

    Techniques Used: DNA Methylation Assay, Methylation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY

    71) Product Images from "Replacement of Glycoprotein B in Alcelaphine Herpesvirus 1 by Its Ovine Herpesvirus 2 Homolog : Implications in Vaccine Development for Sheep-Associated Malignant Catarrhal Fever"

    Article Title: Replacement of Glycoprotein B in Alcelaphine Herpesvirus 1 by Its Ovine Herpesvirus 2 Homolog : Implications in Vaccine Development for Sheep-Associated Malignant Catarrhal Fever

    Journal: mSphere

    doi: 10.1128/mSphere.00108-16

    Reactivity of OvHV-2 gB-specific antibodies to the AlHV-1 ΔORF73/OvHV-2-ORF8 virus. Representative fluorescence microscopy images of FMSK hTERT.1 cells harvested at 24 h posttransfection with AlHV-1 ΔORF73/OvHV-2-ORF8 (A and C), pOvHV-2 ORF8 (B), or AlHV-1 ΔORF73/ΔORF8 (D) DNA and untransfected cells (E). Cells were treated with OvHV-2 gB hyperimmune mouse serum (A, B, D, and E) or preimmune serum (C) as a primary antibody and an anti-mouse IgG conjugated to Alexa Fluor 568 as a secondary antibody. Slides were mounted with SlowFade Gold Antifade Mountant with DAPI and examined using fluorescence microscopy. Individual images from the indicated area of merged image A are shown in A.1, A.2, and A.3. Red fluorescence indicates reactivity of serum antibodies with gB, green fluorescence indicates expression of green fluorescent protein encoded by the BAC cassette, and cell nuclei are stained blue. Magnification, ×20.
    Figure Legend Snippet: Reactivity of OvHV-2 gB-specific antibodies to the AlHV-1 ΔORF73/OvHV-2-ORF8 virus. Representative fluorescence microscopy images of FMSK hTERT.1 cells harvested at 24 h posttransfection with AlHV-1 ΔORF73/OvHV-2-ORF8 (A and C), pOvHV-2 ORF8 (B), or AlHV-1 ΔORF73/ΔORF8 (D) DNA and untransfected cells (E). Cells were treated with OvHV-2 gB hyperimmune mouse serum (A, B, D, and E) or preimmune serum (C) as a primary antibody and an anti-mouse IgG conjugated to Alexa Fluor 568 as a secondary antibody. Slides were mounted with SlowFade Gold Antifade Mountant with DAPI and examined using fluorescence microscopy. Individual images from the indicated area of merged image A are shown in A.1, A.2, and A.3. Red fluorescence indicates reactivity of serum antibodies with gB, green fluorescence indicates expression of green fluorescent protein encoded by the BAC cassette, and cell nuclei are stained blue. Magnification, ×20.

    Techniques Used: Fluorescence, Microscopy, Expressing, BAC Assay, Staining

    Plaque formation and viral replication in cell culture. (A) Representative fluorescence microscopy images of FMSK hTERT.1 cells transfected with AlHV-1 BAC DNA from different constructs at 24 and 96 h posttransfection. Nontransfected cells (mock) were used as a control. Virus spreading and cytopathic effect are indicated by the formation of plaques. Green fluorescence indicates expression of green fluorescent protein encoded by the BAC cassette. BF, bright field; F, fluorescence with a fluorescein isothiocyanate (FITC) filter. Magnification, ×10. (B) Representative images of FMSK hTERT.1 cells infected with AlHV-1 ΔORF73/OvHV-2-ORF8 reconstituted from FMSK hTERT.1 (BAC + , BAC cassette intact) or FMSK hTERT.1 /Cre cells (BAC − , BAC cassette excised) at 96 h postinfection.
    Figure Legend Snippet: Plaque formation and viral replication in cell culture. (A) Representative fluorescence microscopy images of FMSK hTERT.1 cells transfected with AlHV-1 BAC DNA from different constructs at 24 and 96 h posttransfection. Nontransfected cells (mock) were used as a control. Virus spreading and cytopathic effect are indicated by the formation of plaques. Green fluorescence indicates expression of green fluorescent protein encoded by the BAC cassette. BF, bright field; F, fluorescence with a fluorescein isothiocyanate (FITC) filter. Magnification, ×10. (B) Representative images of FMSK hTERT.1 cells infected with AlHV-1 ΔORF73/OvHV-2-ORF8 reconstituted from FMSK hTERT.1 (BAC + , BAC cassette intact) or FMSK hTERT.1 /Cre cells (BAC − , BAC cassette excised) at 96 h postinfection.

    Techniques Used: Cell Culture, Fluorescence, Microscopy, Transfection, BAC Assay, Construct, Expressing, Infection

    72) Product Images from "Poly (ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress-granule localization"

    Article Title: Poly (ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress-granule localization

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.07.002

    The PAR-binding region and the N-terminus are required for TDP-43 LLPS in vitro . (A) The NLS of human TDP-43 and the Drosophila homologue TAR DNA-binding protein-43 homolog (TBPH), share 42.9% identity. The protein domains of TDP-43-WT, -Q331K, ΔPBM, -C35 (amino acids 85–414) and -C25 (amino acids 176–414) are shown. NLS: nuclear localization sequence, PBM: PAR-binding motif, RRM: RNA recognition motif, NES: nuclear export sequence and PrLD: prion-like domain. (B) LLPS of 10 μM protein in 150mM NaCl. SUMO-TDP-43-WT underwent LLPS but SUMOTDP-43-ΔPBM and SUMO-TDP-43-C35 formed irregular-solid structures that did not fuse. SUMO-TDP-43-C25 formed fibrillar aggregates. TDP-43-Q331K formed liquid droplets and the occasional irregular-solid structure. Performed three independent times with two protein preparations of TDP-43-WT, C35 and C25 and twice from one protein preparation of TDP-43-Q331K. (C) The presence of liquid droplets, no liquid droplet, irregular solid structures, both liquid droplets and irregular solid structures and fibrillar aggregates is plotted. (D) LLPS at 5 μM protein and 50mM NaCl. PAR at 5 μM equivalents to ADP-ribose promoted LLPS of TDP-43-WT and did not promote LLPS of TDP-43-ΔPBM, TDP-43-C35, and TDP-43-C25. PAR promoted the formation of irregular-solid structures of TDP-43Q331K. Performed three independent times with two independent protein preparations. (E) The presence of liquid droplets, no liquid droplet, irregular-solid structures and fibrillar aggregates is plotted for each TDP-43 variant in the absence and presence of PAR.All at room temperature.
    Figure Legend Snippet: The PAR-binding region and the N-terminus are required for TDP-43 LLPS in vitro . (A) The NLS of human TDP-43 and the Drosophila homologue TAR DNA-binding protein-43 homolog (TBPH), share 42.9% identity. The protein domains of TDP-43-WT, -Q331K, ΔPBM, -C35 (amino acids 85–414) and -C25 (amino acids 176–414) are shown. NLS: nuclear localization sequence, PBM: PAR-binding motif, RRM: RNA recognition motif, NES: nuclear export sequence and PrLD: prion-like domain. (B) LLPS of 10 μM protein in 150mM NaCl. SUMO-TDP-43-WT underwent LLPS but SUMOTDP-43-ΔPBM and SUMO-TDP-43-C35 formed irregular-solid structures that did not fuse. SUMO-TDP-43-C25 formed fibrillar aggregates. TDP-43-Q331K formed liquid droplets and the occasional irregular-solid structure. Performed three independent times with two protein preparations of TDP-43-WT, C35 and C25 and twice from one protein preparation of TDP-43-Q331K. (C) The presence of liquid droplets, no liquid droplet, irregular solid structures, both liquid droplets and irregular solid structures and fibrillar aggregates is plotted. (D) LLPS at 5 μM protein and 50mM NaCl. PAR at 5 μM equivalents to ADP-ribose promoted LLPS of TDP-43-WT and did not promote LLPS of TDP-43-ΔPBM, TDP-43-C35, and TDP-43-C25. PAR promoted the formation of irregular-solid structures of TDP-43Q331K. Performed three independent times with two independent protein preparations. (E) The presence of liquid droplets, no liquid droplet, irregular-solid structures and fibrillar aggregates is plotted for each TDP-43 variant in the absence and presence of PAR.All at room temperature.

    Techniques Used: Binding Assay, In Vitro, Sequencing, Variant Assay

    73) Product Images from "Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System"

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158889

    Restriction patterns of DNAs : isolated from viral particles (a mixture of ФAH14a and ФAH14b genomic DNA) cleaved with selected REases: HpaII (CCGG), MspI (CCGG) and SmaI (CCCGGG) [panel A] and Pseudomonas sp. ANT_H14 genomic DNA cleaved with SmaI (CCCGGG) [panel B]. Digest mixtures were electrophoresed on 0.8% agarose gels and stained with ethidium bromide. ND–undigested DNA isolated from viral particles; λ/S–DNA of λ dam - dcm - digested with SmaI; Ps–undigested Pseudomonas sp. ANT_H14 genomic DNA; Ps/S–ANT_H14 genomic DNA digested with SmaI; M–GeneRuler 100–10,000 bp size marker.
    Figure Legend Snippet: Restriction patterns of DNAs : isolated from viral particles (a mixture of ФAH14a and ФAH14b genomic DNA) cleaved with selected REases: HpaII (CCGG), MspI (CCGG) and SmaI (CCCGGG) [panel A] and Pseudomonas sp. ANT_H14 genomic DNA cleaved with SmaI (CCCGGG) [panel B]. Digest mixtures were electrophoresed on 0.8% agarose gels and stained with ethidium bromide. ND–undigested DNA isolated from viral particles; λ/S–DNA of λ dam - dcm - digested with SmaI; Ps–undigested Pseudomonas sp. ANT_H14 genomic DNA; Ps/S–ANT_H14 genomic DNA digested with SmaI; M–GeneRuler 100–10,000 bp size marker.

    Techniques Used: Isolation, Staining, Marker

    74) Product Images from "Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii"

    Article Title: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03991-6

    EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
    Figure Legend Snippet: EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different

    Techniques Used: Isolation, Infection, Activation Assay, Incubation

    75) Product Images from "Persistent Viral Reservoirs in Lymphoid Tissues in SIV-Infected Rhesus Macaques of Chinese-Origin on Suppressive Antiretroviral Therapy"

    Article Title: Persistent Viral Reservoirs in Lymphoid Tissues in SIV-Infected Rhesus Macaques of Chinese-Origin on Suppressive Antiretroviral Therapy

    Journal: Viruses

    doi: 10.3390/v11020105

    Levels of cell-associated SIV DNA ( A ) and RNA ( B ) in blood, axillary lymph nodes (AxiLNs), inguinal lymph nodes (IngLNs), mesenteric lymph nodes (MesLNs) and the spleen at the end of 6 months of ART in four animals with full viral suppression in peripheral blood. The dotted line indicates the limit of detection.
    Figure Legend Snippet: Levels of cell-associated SIV DNA ( A ) and RNA ( B ) in blood, axillary lymph nodes (AxiLNs), inguinal lymph nodes (IngLNs), mesenteric lymph nodes (MesLNs) and the spleen at the end of 6 months of ART in four animals with full viral suppression in peripheral blood. The dotted line indicates the limit of detection.

    Techniques Used:

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    Construct:

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    SYBR Green Assay:

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    Expressing:

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: For experiments performed to investigate regional differences in gene expression, the olfactory tissue anterior to the olfactory turbinate was excised from the posterior olfactory tissue containing turbinate and posterior dorsal recess, and total RNA was separately extracted from both regions. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s instructions. .. Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific).

    Article Title: Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression
    Article Snippet: .. M, Gene ruler expression ladder (SM1551, Life technology). ..

    BIA-KA:

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). .. Reagents used for cell proliferation, migration and adhesion assays were as follows: Bicinchoninic acid (BCA) kit (P0011, Beyotime Institute of Biotechnology); Cell Counting Kit-8 (CCK-8; CK04, Dojindo Molecular Technologies Inc., Rockville, MD, USA); Transwell plate (8 µm pore width; Corning Inc.); crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); 0.05% trypsin (Invitrogen; Thermo Fisher Scientific Inc.).

    Ex Vivo:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Paragraph title: Supporting Information BLI signal from ex vivo organs of individual female (n = 8) and male mice (n = 6) infected with T . b . gambiense 1135 for 12–18 months. BLI signal from ex vivo organs of individual females (n = 8) crossed with T . b . gambiense 1135 infected males examined seven months post-crossing. BLI signal from ex vivo organs of male (n = 5) and female (n = 5) offspring (2–3 months old) of T . b . gambiense 1135 infected females (n = 10) crossed with healthy males (n = 5). Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc). Investigation of the vertical transmission of T . b . gambiense 1135 (Rluc). Primer sequences used in the study for the sensitive and specific detection of very low parasitaemia. Fold increase of BLI signal to control from ex-vivo organs of T . b . gambiense 1135 infected male (n = 6) and female mice (n = 8) 12–18 months post-infection. Investigation of horizontal transmission of T . b . gambiense 1135 from infected male mice (n = 5) to healthy female mice (n = 8) examined by ex-vivo organ BLI and PCR seven months post-crossing. Fold increase of BLI signal to control for five female and five male offspring of T . b . gambiense 1135 infected females (n = 10) and healthy male mice (n = 5) examined at 2–3 months of age. ... Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Sequencing:

    Article Title: Detection of novel 3' untranslated region extensions with 3' expression microarrays
    Article Snippet: Fragments were separated on a 1.5% agarose gel and visualized with SYBRSafe dye (Invitrogen), together with a Generuler DNA ladder (Fermentas). .. PCR products for Olfml1 and Riok2 were cut out from the gel, purified and sent out for sequencing to Makrogen.

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: Paragraph title: Sequencing and sequence analysis ... Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany).

    Southern Blot:

    Article Title: Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum
    Article Snippet: Validation of homologous integration within the recipient strain ∆ku70 FRT2 was performed by Southern blotting of 20 μg Eco RV digested genomic DNA with 32 P-labled complementary DNA probes of the 5'- (dark bar) and 3'-flank (bright bar) of dcl2 . .. GeneRuler DNA Ladder (Thermo Scientific) was used as size standard.

    Serial Dilution:

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: .. PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . ..

    Biomarker Assay:

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . .. Replication genotyping was performed using samples obtained though AddNeuromed, a cross European, public/private consortium developed for AD biomarker discovery and a UK cohort from the Medical Research Council Genetic Resource for Late-onset AD .

    Infection:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Paragraph title: Supporting Information BLI signal from ex vivo organs of individual female (n = 8) and male mice (n = 6) infected with T . b . gambiense 1135 for 12–18 months. BLI signal from ex vivo organs of individual females (n = 8) crossed with T . b . gambiense 1135 infected males examined seven months post-crossing. BLI signal from ex vivo organs of male (n = 5) and female (n = 5) offspring (2–3 months old) of T . b . gambiense 1135 infected females (n = 10) crossed with healthy males (n = 5). Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc). Investigation of the vertical transmission of T . b . gambiense 1135 (Rluc). Primer sequences used in the study for the sensitive and specific detection of very low parasitaemia. Fold increase of BLI signal to control from ex-vivo organs of T . b . gambiense 1135 infected male (n = 6) and female mice (n = 8) 12–18 months post-infection. Investigation of horizontal transmission of T . b . gambiense 1135 from infected male mice (n = 5) to healthy female mice (n = 8) examined by ex-vivo organ BLI and PCR seven months post-crossing. Fold increase of BLI signal to control for five female and five male offspring of T . b . gambiense 1135 infected females (n = 10) and healthy male mice (n = 5) examined at 2–3 months of age. ... Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Generated:

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: Sequencing and sequence analysis Complementary cDNA was generated from 4 μl RNA using Omniscript RT Kit (Qiagen, Hilden, Germany) along with a primer specific to the conserved 12 nucleotide of 3’end of the viral RNA as previously published [ ]. .. Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany).

    Transmission Assay:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Paragraph title: Supporting Information BLI signal from ex vivo organs of individual female (n = 8) and male mice (n = 6) infected with T . b . gambiense 1135 for 12–18 months. BLI signal from ex vivo organs of individual females (n = 8) crossed with T . b . gambiense 1135 infected males examined seven months post-crossing. BLI signal from ex vivo organs of male (n = 5) and female (n = 5) offspring (2–3 months old) of T . b . gambiense 1135 infected females (n = 10) crossed with healthy males (n = 5). Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc). Investigation of the vertical transmission of T . b . gambiense 1135 (Rluc). Primer sequences used in the study for the sensitive and specific detection of very low parasitaemia. Fold increase of BLI signal to control from ex-vivo organs of T . b . gambiense 1135 infected male (n = 6) and female mice (n = 8) 12–18 months post-infection. Investigation of horizontal transmission of T . b . gambiense 1135 from infected male mice (n = 5) to healthy female mice (n = 8) examined by ex-vivo organ BLI and PCR seven months post-crossing. Fold increase of BLI signal to control for five female and five male offspring of T . b . gambiense 1135 infected females (n = 10) and healthy male mice (n = 5) examined at 2–3 months of age. ... Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Polymerase Chain Reaction:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Paragraph title: Supporting Information BLI signal from ex vivo organs of individual female (n = 8) and male mice (n = 6) infected with T . b . gambiense 1135 for 12–18 months. BLI signal from ex vivo organs of individual females (n = 8) crossed with T . b . gambiense 1135 infected males examined seven months post-crossing. BLI signal from ex vivo organs of male (n = 5) and female (n = 5) offspring (2–3 months old) of T . b . gambiense 1135 infected females (n = 10) crossed with healthy males (n = 5). Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc). Investigation of the vertical transmission of T . b . gambiense 1135 (Rluc). Primer sequences used in the study for the sensitive and specific detection of very low parasitaemia. Fold increase of BLI signal to control from ex-vivo organs of T . b . gambiense 1135 infected male (n = 6) and female mice (n = 8) 12–18 months post-infection. Investigation of horizontal transmission of T . b . gambiense 1135 from infected male mice (n = 5) to healthy female mice (n = 8) examined by ex-vivo organ BLI and PCR seven months post-crossing. Fold increase of BLI signal to control for five female and five male offspring of T . b . gambiense 1135 infected females (n = 10) and healthy male mice (n = 5) examined at 2–3 months of age. ... Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific). .. Amplicons were then extracted from the agarose gel using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel) before being ligated into the pGEMT-Easy vector (Promega, Fitchburg, WI, USA) to prepare the in situ hybridization riboprobes.

    Article Title: Development of primary cell culture from Scylla serrata
    Article Snippet: Paragraph title: Genomic deoxyribonucleic acid analysis by polymerase chain reaction ... Molecular weight marker, DNA ladder, was from Fermentas (Cat. No. SM1553).

    Article Title: Detection of novel 3' untranslated region extensions with 3' expression microarrays
    Article Snippet: Thermal cycling was performed on a GeneAmp PCR System 9700 (Applied Biosystems), 2' 94°C - 38 × (20" 94°C - 30" 60°C - 3' 72°C) - 10' 72°C - 4°C. .. Fragments were separated on a 1.5% agarose gel and visualized with SYBRSafe dye (Invitrogen), together with a Generuler DNA ladder (Fermentas).

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: All PCR reactions were performed in Thermocycler machine (Eppendorf, Hamburg, Germany) as previously done [ ]: an initial denaturation step (98 °C 30 s), followed by 35 cycles each consisting of 98 °C 10s, 60 °C 30s, 72 °C 6 min and final elongation (72 °C 5 min) utilizing 2 U Phusion High-Fidelity DNA Polymerase (New England BioLabs, Frankfurt am Main, Germany) according to the manufacturer’s guidelines. .. Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany).

    Article Title: Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum
    Article Snippet: In contrast to the tested transformants, the no-template controls (-Control) and PCRs with the recipient DNA (WT) show no PCR product. .. GeneRuler DNA Ladder (Thermo Scientific) was used as size standard.

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish
    Article Snippet: Paragraph title: Screening F1s for loxP integrations by short flanking PCR ... DNA ladders used were Thermo Scientific GeneRuler DNA ladder.

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: .. PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . ..

    Molecular Weight:

    Article Title: Development of primary cell culture from Scylla serrata
    Article Snippet: .. Molecular weight marker, DNA ladder, was from Fermentas (Cat. No. SM1553). .. A cell isolation procedure for Scylla serrata hepatopancreas was developed based on mechanical dissociation and centrifugation method which produced viable cell population.

    RNA Sequencing Assay:

    Article Title: Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum
    Article Snippet: Paragraph title: Supporting Information Annotation of small RNA loci of all three datasets, RNA-Mix, ∆ku70 FRT2, and ∆dcl2 ∆dcl1 . Nucleotide preference of Dicer-dependent and-independent small RNAs. Comparative analysis of the Dicer-protein domains of DCL-2 and DCL-1 in N . crassa and Dcl2 (Pc12g13700) and Dcl1 (Pc21g06890) in P . chrysogenum . Construction and validation of ∆dcl2 mutants. Construction and validation of ∆dcl1 mutants. Construction of ∆dcl2 ∆dcl1 double mutants based on the strain ∆dcl2 . Detailed list of predicted milRNA target sites. Strains used in this study. Cultivations used for RNA-Seq samples. Components involved in small RNA biogenesis of Penicillium chrysogenum compared to five other fungi. List of plasmids used in this study. List of oligonucleotides used in this study. ... GeneRuler DNA Ladder (Thermo Scientific) was used as size standard.

    Fluorescence:

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: .. PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . ..

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: .. Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). ..

    Mouse Assay:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Paragraph title: Supporting Information BLI signal from ex vivo organs of individual female (n = 8) and male mice (n = 6) infected with T . b . gambiense 1135 for 12–18 months. BLI signal from ex vivo organs of individual females (n = 8) crossed with T . b . gambiense 1135 infected males examined seven months post-crossing. BLI signal from ex vivo organs of male (n = 5) and female (n = 5) offspring (2–3 months old) of T . b . gambiense 1135 infected females (n = 10) crossed with healthy males (n = 5). Investigation of the horizontal transmission of T . b . gambiense 1135 (Rluc). Investigation of the vertical transmission of T . b . gambiense 1135 (Rluc). Primer sequences used in the study for the sensitive and specific detection of very low parasitaemia. Fold increase of BLI signal to control from ex-vivo organs of T . b . gambiense 1135 infected male (n = 6) and female mice (n = 8) 12–18 months post-infection. Investigation of horizontal transmission of T . b . gambiense 1135 from infected male mice (n = 5) to healthy female mice (n = 8) examined by ex-vivo organ BLI and PCR seven months post-crossing. Fold increase of BLI signal to control for five female and five male offspring of T . b . gambiense 1135 infected females (n = 10) and healthy male mice (n = 5) examined at 2–3 months of age. ... Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Article Title: Detection of novel 3' untranslated region extensions with 3' expression microarrays
    Article Snippet: PCR was performed in a 25 μl volume with 0.2 μl Platinum Taq (Invitrogen) in Platinum Taq buffer, 300 nM primers (Sigma-Aldrich), 5 ng cDNA, 200 μM nucleotide mix (Fermentas) and 1.5 mM MgCl2 (Invitrogen). cDNA was prepared from the liver and gastrocnemius muscle of male C57Bl6 mice. .. Fragments were separated on a 1.5% agarose gel and visualized with SYBRSafe dye (Invitrogen), together with a Generuler DNA ladder (Fermentas).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: A 1-μL aliquot of this cDNA was then used for each NT and NTR RT-PCR run. .. Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific).

    Article Title: Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression
    Article Snippet: B. RT-PCR detected the expression of short fragment of KRT7-AS RNA from the antisense-specific strand reverse-transcribed cDNA. .. M, Gene ruler expression ladder (SM1551, Life technology).

    Staining:

    Article Title: Development of primary cell culture from Scylla serrata
    Article Snippet: The resulting products were electrophoretically analyzed through 1.6% agarose gels, stained with ethidium bromide, and visualized using a UV transilluminator. .. Molecular weight marker, DNA ladder, was from Fermentas (Cat. No. SM1553).

    Nested PCR:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Respectively lane 1 to 11: 1-nested PCR of the first PCR negative control (water); 2-nested PCR of the first PCR positive control (1 ng T . b . gambiense DNA); 3-nested PCR of the first PCR negative control (1 ng T . congolense DNA); 4-negative control (water) of the nested PCR; 5-positive control (1 ng T . b . gambiense DNA) of the nested PCR; 6-negative control (1 ng T . congolense DNA) of the nested PCR; 7-healty mouse blood extract; 8-T. b. gambiense 1135 mouse blood extract; 9-negative control (water) of the first PCR; 10-positive control (1 ng T . b . gambiense DNA) of the first PCR; 11-negative control (1 ng T . congolense DNA) of the first PCR. .. Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Purification:

    Article Title: Detection of novel 3' untranslated region extensions with 3' expression microarrays
    Article Snippet: Fragments were separated on a 1.5% agarose gel and visualized with SYBRSafe dye (Invitrogen), together with a Generuler DNA ladder (Fermentas). .. PCR products for Olfml1 and Riok2 were cut out from the gel, purified and sent out for sequencing to Makrogen.

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany). .. Amplicons were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish
    Article Snippet: PCRs were run on 2% agarose gels, and amplicons of appropriate size were purified and sequenced to confirm loxP integration. .. DNA ladders used were Thermo Scientific GeneRuler DNA ladder.

    In Situ Hybridization:

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific). .. Amplicons were then extracted from the agarose gel using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel) before being ligated into the pGEMT-Easy vector (Promega, Fitchburg, WI, USA) to prepare the in situ hybridization riboprobes.

    Plasmid Preparation:

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific). .. Amplicons were then extracted from the agarose gel using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel) before being ligated into the pGEMT-Easy vector (Promega, Fitchburg, WI, USA) to prepare the in situ hybridization riboprobes.

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: .. Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). ..

    Software:

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: .. PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . ..

    Real-time Polymerase Chain Reaction:

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: Paragraph title: 4.4. Real-Time Quantitative PCR ... Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific).

    Negative Control:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: Respectively lane 1 to 11: 1-nested PCR of the first PCR negative control (water); 2-nested PCR of the first PCR positive control (1 ng T . b . gambiense DNA); 3-nested PCR of the first PCR negative control (1 ng T . congolense DNA); 4-negative control (water) of the nested PCR; 5-positive control (1 ng T . b . gambiense DNA) of the nested PCR; 6-negative control (1 ng T . congolense DNA) of the nested PCR; 7-healty mouse blood extract; 8-T. b. gambiense 1135 mouse blood extract; 9-negative control (water) of the first PCR; 10-positive control (1 ng T . b . gambiense DNA) of the first PCR; 11-negative control (1 ng T . congolense DNA) of the first PCR. .. Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Agarose Gel Electrophoresis:

    Article Title: Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission
    Article Snippet: 1.8% agarose gel of PCR using Tbingi-F1/pMUTec-R2 nested primers on the blood of 8 female mice taken 3.5, 5 and 6 months post-crossing with T . b . gambiense 1135(Rluc) infected male. .. Lane M: GeneRuler DNA ladder (Thermo Scientific). (DOCX) Click here for additional data file.

    Article Title: Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells
    Article Snippet: .. Amplicons were then run on a 2% agarose gel and the expected sizes were determined by comparing amplicons with GeneRuler DNA ladder (1 kb or/and 1 kb plus; Thermo Fisher Scientific). .. Amplicons were then extracted from the agarose gel using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel) before being ligated into the pGEMT-Easy vector (Promega, Fitchburg, WI, USA) to prepare the in situ hybridization riboprobes.

    Article Title: Detection of novel 3' untranslated region extensions with 3' expression microarrays
    Article Snippet: .. Fragments were separated on a 1.5% agarose gel and visualized with SYBRSafe dye (Invitrogen), together with a Generuler DNA ladder (Fermentas). .. PCR products for Olfml1 and Riok2 were cut out from the gel, purified and sent out for sequencing to Makrogen.

    Electrophoresis:

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: .. Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany). .. Amplicons were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).

    CCK-8 Assay:

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). .. Reagents used for cell proliferation, migration and adhesion assays were as follows: Bicinchoninic acid (BCA) kit (P0011, Beyotime Institute of Biotechnology); Cell Counting Kit-8 (CCK-8; CK04, Dojindo Molecular Technologies Inc., Rockville, MD, USA); Transwell plate (8 µm pore width; Corning Inc.); crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); 0.05% trypsin (Invitrogen; Thermo Fisher Scientific Inc.).

    Knock-Out:

    Article Title: Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum
    Article Snippet: GeneRuler DNA Ladder (Thermo Scientific) was used as size standard. .. Homologous integration of the dcl1 knockout construct resulted in the replacement of the dlc1 coding gene (Pc21g06890) with a nourseothricin resistance cassette.

    Concentration Assay:

    Article Title: Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease
    Article Snippet: .. PCR product concentration was estimated by comparison of the level of fluorescence to a serial dilution of Generuler DNA ladder (Fermentas Life Sciences) using image J software . ..

    Migration:

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). .. Reagents used for cell proliferation, migration and adhesion assays were as follows: Bicinchoninic acid (BCA) kit (P0011, Beyotime Institute of Biotechnology); Cell Counting Kit-8 (CCK-8; CK04, Dojindo Molecular Technologies Inc., Rockville, MD, USA); Transwell plate (8 µm pore width; Corning Inc.); crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); 0.05% trypsin (Invitrogen; Thermo Fisher Scientific Inc.).

    Marker:

    Article Title: Development of primary cell culture from Scylla serrata
    Article Snippet: .. Molecular weight marker, DNA ladder, was from Fermentas (Cat. No. SM1553). .. A cell isolation procedure for Scylla serrata hepatopancreas was developed based on mechanical dissociation and centrifugation method which produced viable cell population.

    Gel Extraction:

    Article Title: Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt
    Article Snippet: Fragments’ sizes were determined by electrophoresis in 1% agar gel in comparison to the GeneRuler™ DNA ladder (Thermo Scientific, Germany). .. Amplicons were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).

    Article Title: Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells
    Article Snippet: .. Reagents for vector construction and identification were as follows: Endonuclease Eco RI/ Bam HI, T4 DNA ligase, GeneRuler DNA ladder (Fermentas; Thermo Fisher Scientific Inc.); primer Oligo, 0.05% Trypsin, Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific Inc.); gel extraction kit (AP-GX-50), plasmid extraction kit (AP-MN-P-50; Corning Inc., New York, NY, USA); packaging mix, 293T cells, cloning vector pcDNA3.1(+)/enhanced green fluorescence protein (EGFP)/internal ribosome entry site (IRES), lentiviral vector pL/GFP/IRES/MCS-BSD (Novobio Inc., Shanghai, China); Escherichia coli ( E. coli ) DH5α competent cells (Takara Biotechnology Inc., Dalian, China). ..

    Fluorescence In Situ Hybridization:

    Article Title: Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish
    Article Snippet: Adult F1 fish were tail clipped, DNA prepped, and genotyped by running a short flanking PCR (gene specific primers described below) designed to amplify approximately 450 bp or less. .. DNA ladders used were Thermo Scientific GeneRuler DNA ladder.

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    The GeneArt Gene Synthesis service offers chemical synthesis cloning and sequence verification of virtually any desired genetic sequence
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    94
    Thermo Fisher prkdc dna pkcs
    <t>DNA</t> repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors <t>DNA-PKcs</t> <t>(Prkdc),</t> DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p
    Prkdc Dna Pkcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher gal4 dna binding domain
    Arabidopsis VPS38 interacts with other subunits of class-III PtdIns-3 kinase complex. (A) Pairwise Y2H assays using the <t>GAL4-based</t> system showing that VPS38 interacts with ATG6. VSPS38 fused to the N terminus of the <t>DNA-binding</t> domain (BD) and ATG6 fused to the N terminus of the activation domain (AD) were co-expressed in yeast and tested for binding by growth on synthetic complete medium lacking leucine, tryptophan, and histidine (-L-W-H), and containing 3-amino-1,2,4-triazole (+3AT). Viability of the cells was confirmed by growth on medium lacking leucine and tryptophan (-L-W). (B) Pairwise Y2H assays by the split-ubiquitin mating system showing interactions among VPS38, ATG6, VPS15, and VPS34. Each full-length protein was expressed as a fusion to either Cub-PLV as bait or NubG as prey, and co-expressed in diploid yeast cells. Positive interactions were determined by growth of twofold serial dilutions on synthetic complete medium lacking uracil, methionine, leucine, tryptophan, histidine, and adenine (-Ura-M-L-W-H-Ade). The empty NubG and NubWT vectors were used as negative and positive controls, respectively. Viability of the cells was confirmed by growth on synthetic complete medium lacking uracil, methionine, leucine, and tryptophan (-Ura-M-L-W). (C) Pairwise BiFC assays showing the interactions among VPS38, ATG6, VPS15, and VPS34 in planta . Each full-length protein was expressed as a fusion to either N-terminal fragment (nYFP) or C-terminal fragment (cYFP) of YFP and then transiently co-expressed in N. benthamiana leaf epidermal cells. Appearance of the fluorescent signals was observed by confocal microscopic analysis 36 h after infiltration. Scale bar = 20 μm. (D) Schematic of the interactions detected among VPS38, ATG6, VPS15, and VPS34. The arrow thickness is an estimate of binding strength based on all interaction assays. The solid and dashed lines indicate interactions that were demonstrated by both Y2H and BiFC, or by just one of the methods, respectively.
    Gal4 Dna Binding Domain, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher dna coated cellulose beads gst p12
    Recombinant <t>GST-Mo-MLV</t> <t>p12</t> does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and <t>DNA</t> (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Dna Coated Cellulose Beads Gst P12, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors DNA-PKcs (Prkdc), DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p

    Journal: Frontiers in Immunology

    Article Title: DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks

    doi: 10.3389/fimmu.2018.01312

    Figure Lengend Snippet: DNA repair and apoptotic proteins expression in normoxic and hypoxic NH-hematopoietic stem cell (HSC) and DN2 thymocytes. (A) Western blot analysis of H2AX (Ser139) phosphorylation in DN2 cells and NH-HSCs cultured in either normoxia (21% O 2 ) or hypoxia (5% O 2 ) at 0–24 h post 4 Gy irradiation. (B) mRNA expression levels of DNA repair factors DNA-PKcs (Prkdc), DNA Ligase IV (Lig4) and Rad51 in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All values were normalized against β-actin and expressed relative to the NH-HSC normoxic sample. Representative western blots of (C) DNA damage response factors DNA ligase IV and 53BP1; and (D) pro- and anti-apoptotic proteins in NH-HSC and DN2 cells in normoxia (21% O 2 ) and hypoxia (5% O 2 ). All graphs show the average of three biological replicates. (* p

    Article Snippet: Reactions were prepared with TaqMan® gene expression master mix (Thermo Fisher Scientific) using predesigned TaqMan® gene expression assays for amplification of mouse Lig4 (DNA Ligase 4), Prkdc (DNA-PKcs), Rad51, and β-actin (Thermo Fisher Scientific).

    Techniques: Expressing, Western Blot, Cell Culture, Irradiation

    DN2 thymocytes activate a robust DNA damage response following γ-irradiation. Western blot analysis of (A) H2AX (Ser139) phosphorylation (γH2AX—marker of DNA double-strand breaks), p53 stabilization; and p21 and Puma expression in DN2 cells and NH-hematopoietic stem cells (HSCs) at 0–24 h post 4 Gy irradiation. Western blot analysis of total endogenous levels of (B) of H2AX, ATM, DNA-PKcs and Chk2; (C) of 53BP1, DNA ligase IV and Rad51; and (D) of Bcl-2, Bcl-XL, and Bim in un-irradiated (control) DN2 cells and NH-HSCs. β-Actin and β-tubulin were used as internal controls. All images are representative of one of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks

    doi: 10.3389/fimmu.2018.01312

    Figure Lengend Snippet: DN2 thymocytes activate a robust DNA damage response following γ-irradiation. Western blot analysis of (A) H2AX (Ser139) phosphorylation (γH2AX—marker of DNA double-strand breaks), p53 stabilization; and p21 and Puma expression in DN2 cells and NH-hematopoietic stem cells (HSCs) at 0–24 h post 4 Gy irradiation. Western blot analysis of total endogenous levels of (B) of H2AX, ATM, DNA-PKcs and Chk2; (C) of 53BP1, DNA ligase IV and Rad51; and (D) of Bcl-2, Bcl-XL, and Bim in un-irradiated (control) DN2 cells and NH-HSCs. β-Actin and β-tubulin were used as internal controls. All images are representative of one of three independent experiments.

    Article Snippet: Reactions were prepared with TaqMan® gene expression master mix (Thermo Fisher Scientific) using predesigned TaqMan® gene expression assays for amplification of mouse Lig4 (DNA Ligase 4), Prkdc (DNA-PKcs), Rad51, and β-actin (Thermo Fisher Scientific).

    Techniques: Irradiation, Western Blot, Marker, Expressing

    Arabidopsis VPS38 interacts with other subunits of class-III PtdIns-3 kinase complex. (A) Pairwise Y2H assays using the GAL4-based system showing that VPS38 interacts with ATG6. VSPS38 fused to the N terminus of the DNA-binding domain (BD) and ATG6 fused to the N terminus of the activation domain (AD) were co-expressed in yeast and tested for binding by growth on synthetic complete medium lacking leucine, tryptophan, and histidine (-L-W-H), and containing 3-amino-1,2,4-triazole (+3AT). Viability of the cells was confirmed by growth on medium lacking leucine and tryptophan (-L-W). (B) Pairwise Y2H assays by the split-ubiquitin mating system showing interactions among VPS38, ATG6, VPS15, and VPS34. Each full-length protein was expressed as a fusion to either Cub-PLV as bait or NubG as prey, and co-expressed in diploid yeast cells. Positive interactions were determined by growth of twofold serial dilutions on synthetic complete medium lacking uracil, methionine, leucine, tryptophan, histidine, and adenine (-Ura-M-L-W-H-Ade). The empty NubG and NubWT vectors were used as negative and positive controls, respectively. Viability of the cells was confirmed by growth on synthetic complete medium lacking uracil, methionine, leucine, and tryptophan (-Ura-M-L-W). (C) Pairwise BiFC assays showing the interactions among VPS38, ATG6, VPS15, and VPS34 in planta . Each full-length protein was expressed as a fusion to either N-terminal fragment (nYFP) or C-terminal fragment (cYFP) of YFP and then transiently co-expressed in N. benthamiana leaf epidermal cells. Appearance of the fluorescent signals was observed by confocal microscopic analysis 36 h after infiltration. Scale bar = 20 μm. (D) Schematic of the interactions detected among VPS38, ATG6, VPS15, and VPS34. The arrow thickness is an estimate of binding strength based on all interaction assays. The solid and dashed lines indicate interactions that were demonstrated by both Y2H and BiFC, or by just one of the methods, respectively.

    Journal: Frontiers in Plant Science

    Article Title: The Vacuolar Protein Sorting-38 Subunit of the Arabidopsis Phosphatidylinositol-3-Kinase Complex Plays Critical Roles in Autophagy, Endosome Sorting, and Gravitropism

    doi: 10.3389/fpls.2018.00781

    Figure Lengend Snippet: Arabidopsis VPS38 interacts with other subunits of class-III PtdIns-3 kinase complex. (A) Pairwise Y2H assays using the GAL4-based system showing that VPS38 interacts with ATG6. VSPS38 fused to the N terminus of the DNA-binding domain (BD) and ATG6 fused to the N terminus of the activation domain (AD) were co-expressed in yeast and tested for binding by growth on synthetic complete medium lacking leucine, tryptophan, and histidine (-L-W-H), and containing 3-amino-1,2,4-triazole (+3AT). Viability of the cells was confirmed by growth on medium lacking leucine and tryptophan (-L-W). (B) Pairwise Y2H assays by the split-ubiquitin mating system showing interactions among VPS38, ATG6, VPS15, and VPS34. Each full-length protein was expressed as a fusion to either Cub-PLV as bait or NubG as prey, and co-expressed in diploid yeast cells. Positive interactions were determined by growth of twofold serial dilutions on synthetic complete medium lacking uracil, methionine, leucine, tryptophan, histidine, and adenine (-Ura-M-L-W-H-Ade). The empty NubG and NubWT vectors were used as negative and positive controls, respectively. Viability of the cells was confirmed by growth on synthetic complete medium lacking uracil, methionine, leucine, and tryptophan (-Ura-M-L-W). (C) Pairwise BiFC assays showing the interactions among VPS38, ATG6, VPS15, and VPS34 in planta . Each full-length protein was expressed as a fusion to either N-terminal fragment (nYFP) or C-terminal fragment (cYFP) of YFP and then transiently co-expressed in N. benthamiana leaf epidermal cells. Appearance of the fluorescent signals was observed by confocal microscopic analysis 36 h after infiltration. Scale bar = 20 μm. (D) Schematic of the interactions detected among VPS38, ATG6, VPS15, and VPS34. The arrow thickness is an estimate of binding strength based on all interaction assays. The solid and dashed lines indicate interactions that were demonstrated by both Y2H and BiFC, or by just one of the methods, respectively.

    Article Snippet: These fragments were then recombined in-frame into the pDEST22 plasmid harboring the GAL4 activation domain and into the pDEST32 plasmid harboring the GAL4 DNA-binding domain via Gateway LR reaction (Thermo Fischer Scientific).

    Techniques: Binding Assay, Activation Assay, Bimolecular Fluorescence Complementation Assay

    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Cell Culture

    Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Binding Assay, Recombinant, De-Phosphorylation Assay, Infection

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    CA and p12 co-localise on mitotic chromatin independently of BET-protein binding. HeLa cells synchronised using a double-aphidicolin block were infected with WT Mo-MLV VLPs or mutants carrying p12_mut14 or IN_W390A. 10 h post-infection, the cells were fixed, stained for p12 (anti-p12, red), CA (anti-CA, green) and DNA (DAPI, blue), and analysed by confocal microscopy. (A) Representative images showing WT p12 and CA co-localisation on mitotic chromatin. Bottom panels are enlarged views of boxed regions in top panels. (B) Representative images of cells infected with VLPs carrying p12_WT (top panels) or p12_mut14 (bottom panels). (C) A representative image of cells infected with VLPs carrying IN_W390A, which is deficient in BET protein binding. (D) Infectivity of Mo-MLV VLPs carrying alterations in p12 and/or IN. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying p12_WT, p12_mut14, p12+ h CBS or (p12+ h CBS)_mut14 in combination with IN_WT or BET-binding deficient IN_W390A. Infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: CA and p12 co-localise on mitotic chromatin independently of BET-protein binding. HeLa cells synchronised using a double-aphidicolin block were infected with WT Mo-MLV VLPs or mutants carrying p12_mut14 or IN_W390A. 10 h post-infection, the cells were fixed, stained for p12 (anti-p12, red), CA (anti-CA, green) and DNA (DAPI, blue), and analysed by confocal microscopy. (A) Representative images showing WT p12 and CA co-localisation on mitotic chromatin. Bottom panels are enlarged views of boxed regions in top panels. (B) Representative images of cells infected with VLPs carrying p12_WT (top panels) or p12_mut14 (bottom panels). (C) A representative image of cells infected with VLPs carrying IN_W390A, which is deficient in BET protein binding. (D) Infectivity of Mo-MLV VLPs carrying alterations in p12 and/or IN. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying p12_WT, p12_mut14, p12+ h CBS or (p12+ h CBS)_mut14 in combination with IN_WT or BET-binding deficient IN_W390A. Infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Protein Binding, Blocking Assay, Infection, Staining, Confocal Microscopy, Binding Assay, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: Pull-down assays using DNA-coated cellulose beads GST-p12 and IN-HA were expressed in 293T cells from pCAGGS/GST and pCMV4/HA-derived plasmids respectively, by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging