Structured Review

Millipore dnase i
The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. <t>DNase</t> I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.
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Images

1) Product Images from "Obesity Risk Gene TMEM18 Encodes a Sequence-Specific DNA-Binding Protein"

Article Title: Obesity Risk Gene TMEM18 Encodes a Sequence-Specific DNA-Binding Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025317

The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. DNase I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.
Figure Legend Snippet: The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. DNase I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.

Techniques Used: Binding Assay, Western Blot, Flow Cytometry

2) Product Images from "The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells"

Article Title: The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0721-8

Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p
Figure Legend Snippet: Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p

Techniques Used: Expressing, Isolation, Mouse Assay, Transplantation Assay, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Luciferase

3) Product Images from "Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells"

Article Title: Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells

Journal: Neuroepigenetics

doi: 10.1016/j.nepig.2016.03.001

To compare Benzonase to DNase I: SH-SY5Y cells were digested with various concentrations of Benzonase (A) or DNase I (B). qPCR indicated that Benzonase digestion exhibited similar patterns of relative openness to DNase I digestion in 7 distinct regions
Figure Legend Snippet: To compare Benzonase to DNase I: SH-SY5Y cells were digested with various concentrations of Benzonase (A) or DNase I (B). qPCR indicated that Benzonase digestion exhibited similar patterns of relative openness to DNase I digestion in 7 distinct regions

Techniques Used: Real-time Polymerase Chain Reaction

4) Product Images from "Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice, et al. Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice"

Article Title: Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice, et al. Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13751

Either Ggnbp2 or Ggn inactivation inhibits meiotic differentiation of GC‐2spd cells. A,B, Three representative stable GC‐2spd cell clones transfected with Ggnbp2 ( Ggnbp2 −/− ) or Ggn ( Ggn −/− ) CRISPR double nickase knockout plasmids contain no detectable Ggnbp2 (A) or Ggn (B) transcripts and proteins demonstrated by RT‐PCR and Western blot, respectively. C,D, Flow cytometric results reveal that significant reduction in haploid cell number in the absence of either Ggnbp2 or Ggn in GC‐2spd cells. Representative histograms (C) and quantitative data (D) are from three experimental replicates. E, RT‐PCR analyses show a remarkable decrease in mRNA levels of spermatid marker genes Prm2 and Acrv1 , while spermatocyte marker gene Pms2 mRNA levels are increased in both Ggnbp2 −/− and Ggn −/− GC‐2spd cells. The experiments are repeated three times and data are presented as mean ± SEM. (** P
Figure Legend Snippet: Either Ggnbp2 or Ggn inactivation inhibits meiotic differentiation of GC‐2spd cells. A,B, Three representative stable GC‐2spd cell clones transfected with Ggnbp2 ( Ggnbp2 −/− ) or Ggn ( Ggn −/− ) CRISPR double nickase knockout plasmids contain no detectable Ggnbp2 (A) or Ggn (B) transcripts and proteins demonstrated by RT‐PCR and Western blot, respectively. C,D, Flow cytometric results reveal that significant reduction in haploid cell number in the absence of either Ggnbp2 or Ggn in GC‐2spd cells. Representative histograms (C) and quantitative data (D) are from three experimental replicates. E, RT‐PCR analyses show a remarkable decrease in mRNA levels of spermatid marker genes Prm2 and Acrv1 , while spermatocyte marker gene Pms2 mRNA levels are increased in both Ggnbp2 −/− and Ggn −/− GC‐2spd cells. The experiments are repeated three times and data are presented as mean ± SEM. (** P

Techniques Used: Clone Assay, Transfection, CRISPR, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Marker

5) Product Images from "Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1"

Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

Journal: Journal for Immunotherapy of Cancer

doi: 10.1186/s40425-018-0452-5

p38-STAT3 signaling in BMDMs and protein fraction in TRAPs are essential for induction of PD-L1 and IL-10. a BMDMs were exposed to TRAPs (10 μg/ml) at indicated time points. Cell lysates were analyzed for p38, p-p38, STAT3 and p-STAT3 by western blot. GAPDH was used as a loading control. b BMDMs were pretreated with p38 inhibitor SB203580 (3 μM) for 1 h, and then co-incubated with TRAPs (10 μg/ml) for 4 h. Expression of STAT3 and p-STAT3 was detected by western blot. c BMDMs were exposed to SB203580 at described concentrations for 1 h, and followed by incubation with TRAPs (10 μg/ml) for 72 h. PD-L1 expression was determined by flow cytometry, and ( d ) the production of IL-10 was assessed by ELISA. e BMDMs were pretreated with STAT3 inhibitor Stattic (1 and 3 μM) for 1 h, and then stimulated with TRAPs (10 μg/ml) for 72 h. PD-L1 expression and ( f ) IL-10 secretion was determined by flow cytometry and ELISA, respectively. g TRAPs were pretreated with Proteinase K for 2 h at 55 °C, DNase I for 1 h at 37 °C or RNase for 3 h at 37 °C, respectively, followed by incubation with BMDMs for 72 h. PD-L1 was evaluated by flow cytometry, and ( h ) IL-10 was tested by ELISA. Data are shown as mean ± SEM, and are representative of three independent experiments. *** p
Figure Legend Snippet: p38-STAT3 signaling in BMDMs and protein fraction in TRAPs are essential for induction of PD-L1 and IL-10. a BMDMs were exposed to TRAPs (10 μg/ml) at indicated time points. Cell lysates were analyzed for p38, p-p38, STAT3 and p-STAT3 by western blot. GAPDH was used as a loading control. b BMDMs were pretreated with p38 inhibitor SB203580 (3 μM) for 1 h, and then co-incubated with TRAPs (10 μg/ml) for 4 h. Expression of STAT3 and p-STAT3 was detected by western blot. c BMDMs were exposed to SB203580 at described concentrations for 1 h, and followed by incubation with TRAPs (10 μg/ml) for 72 h. PD-L1 expression was determined by flow cytometry, and ( d ) the production of IL-10 was assessed by ELISA. e BMDMs were pretreated with STAT3 inhibitor Stattic (1 and 3 μM) for 1 h, and then stimulated with TRAPs (10 μg/ml) for 72 h. PD-L1 expression and ( f ) IL-10 secretion was determined by flow cytometry and ELISA, respectively. g TRAPs were pretreated with Proteinase K for 2 h at 55 °C, DNase I for 1 h at 37 °C or RNase for 3 h at 37 °C, respectively, followed by incubation with BMDMs for 72 h. PD-L1 was evaluated by flow cytometry, and ( h ) IL-10 was tested by ELISA. Data are shown as mean ± SEM, and are representative of three independent experiments. *** p

Techniques Used: Western Blot, Incubation, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

6) Product Images from "The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori"

Article Title: The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00840

DNase I protection patterns of Fur on the pfr promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the P pfr probe in presence of 150 μM of (NH 4 ) 2 Fe(SO 4 ) 2 (A) or 150 μM Dipyridyl (B) . A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo -Fur operators; white, apo -Fur operators. Arrowheads indicate hypersensitivity bands to DNase I treatment. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H 2 O 2 (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.
Figure Legend Snippet: DNase I protection patterns of Fur on the pfr promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the P pfr probe in presence of 150 μM of (NH 4 ) 2 Fe(SO 4 ) 2 (A) or 150 μM Dipyridyl (B) . A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo -Fur operators; white, apo -Fur operators. Arrowheads indicate hypersensitivity bands to DNase I treatment. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H 2 O 2 (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.

Techniques Used: Footprinting

DNase I protection patterns of Fur on the frpB promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the P pfr probe in presence of 150 μM of (NH 4 ) 2 Fe(SO 4 ) 2 (A) or 150 μM Dipyridyl (B) . A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo -Fur operators; white, apo -Fur operators. Arrowheads indicate hypersensitivity bands to DNase I digestion. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H 2 O 2 (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.
Figure Legend Snippet: DNase I protection patterns of Fur on the frpB promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the P pfr probe in presence of 150 μM of (NH 4 ) 2 Fe(SO 4 ) 2 (A) or 150 μM Dipyridyl (B) . A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo -Fur operators; white, apo -Fur operators. Arrowheads indicate hypersensitivity bands to DNase I digestion. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H 2 O 2 (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.

Techniques Used: Footprinting

7) Product Images from "Neutrophil extracellular traps promote lipopolysaccharide-induced airway inflammation and mucus hypersecretion in mice"

Article Title: Neutrophil extracellular traps promote lipopolysaccharide-induced airway inflammation and mucus hypersecretion in mice

Journal: Oncotarget

doi: 10.18632/oncotarget.24022

Degradation of NETs by aerosolized DNase I decreased LPS-induced airway inflammation in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. The lung tissues and BALF from the mice were collected 24 hours after LPS administration. (A) Representative figures (×200) ofhematoxylin and eosin staining in mouse lung tissues. (B) Total cells, neutrophils, macrophages and lymphocytes in BALF were counted under × 400 magnification using a light microscope. (C) MouseTNF-α, IL-6 and IL-1β levels in BALF were measured using ELISA. Data represent the mean ± SD (n=6). * P
Figure Legend Snippet: Degradation of NETs by aerosolized DNase I decreased LPS-induced airway inflammation in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. The lung tissues and BALF from the mice were collected 24 hours after LPS administration. (A) Representative figures (×200) ofhematoxylin and eosin staining in mouse lung tissues. (B) Total cells, neutrophils, macrophages and lymphocytes in BALF were counted under × 400 magnification using a light microscope. (C) MouseTNF-α, IL-6 and IL-1β levels in BALF were measured using ELISA. Data represent the mean ± SD (n=6). * P

Techniques Used: Mouse Assay, Injection, Staining, Light Microscopy, Enzyme-linked Immunosorbent Assay

LPS-induced NET formation was degraded by aerosolized DNase I in mice Mice received aerosolized DNase I (120 U of DNase I in 5mL of normal saline [NS]) or NS at 4 and 12 hours after LPS injection (2 mg of LPS in 50μL NS). (A) Representative images (×200) show merged neutrophil DNA (blue), Cit-H3 (green), and MPO (red) via confocal microscopy of lung sections from mice in the NS, LPS and LPS plus DNase I groups. Arrows show NET formation. (B, C) Representative Western blots and quantification of Cit-H3 protein expression in lung homogenates of all groups. (D) BALF levels of NET-DNA were detected using an MPO-DNA ELISA kit. * P
Figure Legend Snippet: LPS-induced NET formation was degraded by aerosolized DNase I in mice Mice received aerosolized DNase I (120 U of DNase I in 5mL of normal saline [NS]) or NS at 4 and 12 hours after LPS injection (2 mg of LPS in 50μL NS). (A) Representative images (×200) show merged neutrophil DNA (blue), Cit-H3 (green), and MPO (red) via confocal microscopy of lung sections from mice in the NS, LPS and LPS plus DNase I groups. Arrows show NET formation. (B, C) Representative Western blots and quantification of Cit-H3 protein expression in lung homogenates of all groups. (D) BALF levels of NET-DNA were detected using an MPO-DNA ELISA kit. * P

Techniques Used: Mouse Assay, Injection, Confocal Microscopy, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Degradation of NETs by aerosolized DNase I decreased LPS-induced mucus hypersecretion in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. The lung tissues and BALF from the mice were collected 24 hours after LPS administration. (A) MUC5AC and MUC5B mRNA levels in lung tissue from each group were detected by quantitative-RT-PCR. (B) MUC5AC and MUC5B secretion in BALF was measured by ELISA. (C) Representative figures (×200) of AB-PAS staining in mouse lung tissues. Data represent the mean ± SD (n=6). * P
Figure Legend Snippet: Degradation of NETs by aerosolized DNase I decreased LPS-induced mucus hypersecretion in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. The lung tissues and BALF from the mice were collected 24 hours after LPS administration. (A) MUC5AC and MUC5B mRNA levels in lung tissue from each group were detected by quantitative-RT-PCR. (B) MUC5AC and MUC5B secretion in BALF was measured by ELISA. (C) Representative figures (×200) of AB-PAS staining in mouse lung tissues. Data represent the mean ± SD (n=6). * P

Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

Degradation of NETs by aerosolized DNase I suppressed activation of the TLR4/NF-κB signaling pathway after LPS exposure in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. Lung tissues from the mice were collected 24 hours after LPS administration. (A) Representative Western blots of TLR4, β-actin, NF-κB p65, p-IKB-α, IKB-α, and histone H3 in lung tissue from each group. (B, C, D) Quantification of Western blot data from A. Data represent the mean ± SD (n=6). * P
Figure Legend Snippet: Degradation of NETs by aerosolized DNase I suppressed activation of the TLR4/NF-κB signaling pathway after LPS exposure in mice Mice received aerosolized DNase I (120 U) or NS at 4 and 12 hours after LPS injection. Lung tissues from the mice were collected 24 hours after LPS administration. (A) Representative Western blots of TLR4, β-actin, NF-κB p65, p-IKB-α, IKB-α, and histone H3 in lung tissue from each group. (B, C, D) Quantification of Western blot data from A. Data represent the mean ± SD (n=6). * P

Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot

8) Product Images from "A viral genome packaging ring-ATPase is a flexibly coordinated pentamer"

Article Title: A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Journal: bioRxiv

doi: 10.1101/2021.03.08.434477

Motors with inactive subunits show a reduced propensity to engage DNA and a reduced packaging efficiency.. Schematic of in vitro bulk packaging assay. After allowing the packaging to proceed, the unpackaged DNA was digested by DNase I and the packaged DNA was retrieved from the capsids by digesting the capsid and DNase I with proteinase K. (b) Agarose gel electrophoresis of DNA packaged by motors constituted with increasing numbers of inactive subunits. The WT gp17 and the inactive Q143A mutant gp17 (inactive subunit) were pre-mixed at different ratios and added to the reaction mixtures containing capsids, ATP, and linearized plasmid DNA. (c) Schematic of single-molecule fluorescence assay. The assay is similar to the one in Fig. 1a but performed with increasing % of inactive subunits (black sphere). Representative CCD images of the Cy5 channel, showing the number of Cy5 spots (each containing 1 to n # of packaged DNA) indicate the packaging efficiency with increasing % of inactive subunits. (e) DNA packaging activity quantified from the agarose gel in (b) (cyan squares) and from the Cy5 spots in (d) (red squares). Also shown are predicted activities where the minimum number of active subunits required for packaging is 1 (no coordination; black) to 5 (strict coordination; gray). Values represent means ± std (n = 2–5). (f) Representative single-molecule trajectories of Cy5 photobleaching steps (red), indicating the number of DNA molecules packaged inside the capsid, and Cy3 photobleaching steps (green), indicating the number of inactive subunits in the motor. Inset: Schematic of the assay, similar to the one shown in (c), with inactive subunits (black sphere) labeled with Cy3. (g) Distribution of the counts of packaged DNA molecules from motors reporting 0-3 Cy3 photobleaching events (indicative of the count of inactive subunits). The average # of packaged DNA is indicated by f and the number of trajectories used for each distribution by n. Error bars throughout represent std.The number of trajectories used is indicated by n=#.
Figure Legend Snippet: Motors with inactive subunits show a reduced propensity to engage DNA and a reduced packaging efficiency.. Schematic of in vitro bulk packaging assay. After allowing the packaging to proceed, the unpackaged DNA was digested by DNase I and the packaged DNA was retrieved from the capsids by digesting the capsid and DNase I with proteinase K. (b) Agarose gel electrophoresis of DNA packaged by motors constituted with increasing numbers of inactive subunits. The WT gp17 and the inactive Q143A mutant gp17 (inactive subunit) were pre-mixed at different ratios and added to the reaction mixtures containing capsids, ATP, and linearized plasmid DNA. (c) Schematic of single-molecule fluorescence assay. The assay is similar to the one in Fig. 1a but performed with increasing % of inactive subunits (black sphere). Representative CCD images of the Cy5 channel, showing the number of Cy5 spots (each containing 1 to n # of packaged DNA) indicate the packaging efficiency with increasing % of inactive subunits. (e) DNA packaging activity quantified from the agarose gel in (b) (cyan squares) and from the Cy5 spots in (d) (red squares). Also shown are predicted activities where the minimum number of active subunits required for packaging is 1 (no coordination; black) to 5 (strict coordination; gray). Values represent means ± std (n = 2–5). (f) Representative single-molecule trajectories of Cy5 photobleaching steps (red), indicating the number of DNA molecules packaged inside the capsid, and Cy3 photobleaching steps (green), indicating the number of inactive subunits in the motor. Inset: Schematic of the assay, similar to the one shown in (c), with inactive subunits (black sphere) labeled with Cy3. (g) Distribution of the counts of packaged DNA molecules from motors reporting 0-3 Cy3 photobleaching events (indicative of the count of inactive subunits). The average # of packaged DNA is indicated by f and the number of trajectories used for each distribution by n. Error bars throughout represent std.The number of trajectories used is indicated by n=#.

Techniques Used: In Vitro, Agarose Gel Electrophoresis, Mutagenesis, Plasmid Preparation, Fluorescence, Activity Assay, Labeling

9) Product Images from "The Copper-Inducible cin Operon Encodes an Unusual Methionine-Rich Azurin-Like Protein and a Pre-Q0 Reductase in Pseudomonas putida KT2440 ▿"

Article Title: The Copper-Inducible cin Operon Encodes an Unusual Methionine-Rich Azurin-Like Protein and a Pre-Q0 Reductase in Pseudomonas putida KT2440 ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00377-07

RT-PCR analysis of expression of cinA and cinQ in P. putida KT2440. Electrophoresis was performed on a 2% agarose gel stained with ethidium bromide. Lane bp, 100-bp ladder; lane 1, RT-PCR product from RNAs extracted from CuCl 2 -induced cells (after DNase I treatment), using RT 5′ sense and RT 3′ antisense primers; lane 2, negative control for RT, obtained by PCR amplification of the RT product obtained when the reverse transcriptase was omitted, to verify that no genomic contamination was present in the RNA extract; lane 3, PCR negative control obtained by omitting the genomic DNA from the PCR; lane 4, PCR positive control obtained using genomic DNA from P. putida KT2440 as the template.
Figure Legend Snippet: RT-PCR analysis of expression of cinA and cinQ in P. putida KT2440. Electrophoresis was performed on a 2% agarose gel stained with ethidium bromide. Lane bp, 100-bp ladder; lane 1, RT-PCR product from RNAs extracted from CuCl 2 -induced cells (after DNase I treatment), using RT 5′ sense and RT 3′ antisense primers; lane 2, negative control for RT, obtained by PCR amplification of the RT product obtained when the reverse transcriptase was omitted, to verify that no genomic contamination was present in the RNA extract; lane 3, PCR negative control obtained by omitting the genomic DNA from the PCR; lane 4, PCR positive control obtained using genomic DNA from P. putida KT2440 as the template.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoresis, Agarose Gel Electrophoresis, Staining, Negative Control, Polymerase Chain Reaction, Amplification, Positive Control

10) Product Images from "Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps"

Article Title: Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps

Journal: BMC Cancer

doi: 10.1186/s12885-018-4584-2

NET upregulation of platelet aggregation is mediated by neutrophil DNA and platelet RAGE. Removing DNA from NET supernatant using DNase I treatment prior to exposure to whole blood reversed the treatment effects of NET supernatant on platelet aggregation in human blood ( a , 25.9 ± 2.2 vs. 11.35 ± 0.31, n = 4, p
Figure Legend Snippet: NET upregulation of platelet aggregation is mediated by neutrophil DNA and platelet RAGE. Removing DNA from NET supernatant using DNase I treatment prior to exposure to whole blood reversed the treatment effects of NET supernatant on platelet aggregation in human blood ( a , 25.9 ± 2.2 vs. 11.35 ± 0.31, n = 4, p

Techniques Used:

11) Product Images from "The S. cerevisiae Rrm3p DNA helicase moves with the replication forkand affects replication of allyeast chromosomes"

Article Title: The S. cerevisiae Rrm3p DNA helicase moves with the replication forkand affects replication of allyeast chromosomes

Journal: Genes & Development

doi: 10.1101/gad.1478906

Rrm3p associates with Pol2p during S phase. Protein extracts were prepared from mid-S-phase cultures of strains SDY1 ( A ) and SDY2 ( B ), treated with DNase I (+) or not (−), and immunoprecipitated (IP) with either anti-C-MYC Agarose-conjugated beads or anti-HA Agarose-conjugated beads. Input and IP samples were separated on SDS-PAGE gels, and analyzed by Western blotting. Blots were probed with anti-HA ( top ) or anti-MYC ( bottom ).
Figure Legend Snippet: Rrm3p associates with Pol2p during S phase. Protein extracts were prepared from mid-S-phase cultures of strains SDY1 ( A ) and SDY2 ( B ), treated with DNase I (+) or not (−), and immunoprecipitated (IP) with either anti-C-MYC Agarose-conjugated beads or anti-HA Agarose-conjugated beads. Input and IP samples were separated on SDS-PAGE gels, and analyzed by Western blotting. Blots were probed with anti-HA ( top ) or anti-MYC ( bottom ).

Techniques Used: Immunoprecipitation, SDS Page, Western Blot

12) Product Images from "The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells"

Article Title: The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0721-8

Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p
Figure Legend Snippet: Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p

Techniques Used: Expressing, Isolation, Mouse Assay, Transplantation Assay, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Luciferase

13) Product Images from "Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System"

Article Title: Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.10.3187-3194.2004

RT-PCR analysis of arsC gene expression in Halobacterium sp. strain NRC-1. A 2% agarose gel is shown after electrophoresis and staining with ethidium bromide. Lanes 1 to 7 contain PCR products using arsC primers. Lanes 1, 2, and 3 contain RT-PCR products from RNAs (after DNase I treatment) purified from Halobacterium sp. strain NRC-1 cultured in CM medium, 25 μM As(III), and 10 μM Sb(III), respectively. Arrows indicate the positions of the major RT-PCR products in lanes 2 and 3. Lanes 4, 5, and 6 contain PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, after DNase I treatment but without RT. Lane 7 contains a positive control PCR fragment using genomic DNA from Halobacterium sp. strain NRC-1 as a template. Lanes 8 to 10 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, but with universal 16S rRNA gene primers. Lanes 11 to 13 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, with archaeal 16S rRNA gene primers. Lane 14 contains a 100-bp ladder as a size marker.
Figure Legend Snippet: RT-PCR analysis of arsC gene expression in Halobacterium sp. strain NRC-1. A 2% agarose gel is shown after electrophoresis and staining with ethidium bromide. Lanes 1 to 7 contain PCR products using arsC primers. Lanes 1, 2, and 3 contain RT-PCR products from RNAs (after DNase I treatment) purified from Halobacterium sp. strain NRC-1 cultured in CM medium, 25 μM As(III), and 10 μM Sb(III), respectively. Arrows indicate the positions of the major RT-PCR products in lanes 2 and 3. Lanes 4, 5, and 6 contain PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, after DNase I treatment but without RT. Lane 7 contains a positive control PCR fragment using genomic DNA from Halobacterium sp. strain NRC-1 as a template. Lanes 8 to 10 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, but with universal 16S rRNA gene primers. Lanes 11 to 13 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, with archaeal 16S rRNA gene primers. Lane 14 contains a 100-bp ladder as a size marker.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis, Staining, Polymerase Chain Reaction, Purification, Cell Culture, Positive Control, Marker

14) Product Images from "Directed evolution of protein enzymes using nonhomologous random recombination"

Article Title: Directed evolution of protein enzymes using nonhomologous random recombination

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

doi: 10.1073/pnas.0402202101

Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry
Figure Legend Snippet: Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry

Techniques Used: Ligation

15) Product Images from "Rapid chromatin repression by Aire provides precise control of immune tolerance"

Article Title: Rapid chromatin repression by Aire provides precise control of immune tolerance

Journal: Nature immunology

doi: 10.1038/s41590-017-0032-8

Aire rapidly represses accessibility upon recruitment to chromatin. ( a ) Schematic of inducible CiA system: Recruitment to modified Oct4 locus via rapamycin (Rap)-induced dimerizing of Aire-Frb and Fkbp-Zfhd1 fusion proteins. ( b ) Changes in DNase I hypersensitivity (left, n = 4 independent experiments - circles) at CiA:Oct4 locus upon Rap-induced (15 min) Aire recruitment measured by V5 ChIP (right, n = 3 independent experiments). Mean +/− s.e.m. ( c ) Depiction of APS-1 patient mutations in CARD, SAND and PHD1 domains. ( d ) Western blot of Aire-Frb (and mutants) transgenic expression in CiA system. Representative of two independent experiments. ( e ) Changes in DNase I hypersensitivity (left, n = 4 independent experiments) at CiA:Oct4 locus upon Rap-induced (15 min) WT Aire or variant (key) recruitment measured by V5 ChIP (right, n = 3 independent experiments). Mean +/− s.e.m. Statistical significance for each mutant relative to WT by two-tailed t-test. P values
Figure Legend Snippet: Aire rapidly represses accessibility upon recruitment to chromatin. ( a ) Schematic of inducible CiA system: Recruitment to modified Oct4 locus via rapamycin (Rap)-induced dimerizing of Aire-Frb and Fkbp-Zfhd1 fusion proteins. ( b ) Changes in DNase I hypersensitivity (left, n = 4 independent experiments - circles) at CiA:Oct4 locus upon Rap-induced (15 min) Aire recruitment measured by V5 ChIP (right, n = 3 independent experiments). Mean +/− s.e.m. ( c ) Depiction of APS-1 patient mutations in CARD, SAND and PHD1 domains. ( d ) Western blot of Aire-Frb (and mutants) transgenic expression in CiA system. Representative of two independent experiments. ( e ) Changes in DNase I hypersensitivity (left, n = 4 independent experiments) at CiA:Oct4 locus upon Rap-induced (15 min) WT Aire or variant (key) recruitment measured by V5 ChIP (right, n = 3 independent experiments). Mean +/− s.e.m. Statistical significance for each mutant relative to WT by two-tailed t-test. P values

Techniques Used: Modification, Chromatin Immunoprecipitation, Western Blot, Transgenic Assay, Expressing, Variant Assay, Mutagenesis, Two Tailed Test

16) Product Images from "Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells"

Article Title: Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells

Journal: Oncotarget

doi: 10.18632/oncotarget.26061

a2V deletion in HSCs cause altered recruitment of immune cell populations into the TME After 14 days of implantation, tumors from control and a2V-KO mice were harvested, mechanically disrupted, and digested with Collagenase IV and DNAse I to obtain a single cell suspension. The single cells were enriched for CD45 expression by MACS and subjected to flow-cytometry and counted manually with trypan blue. Representative histograms are shown in the left side. Bar graphs show the number of single, live, CD45 + cells per gram of tumor in the TME. (A) MDSC (B) T cells (C) αβ T cells and γδ T cells (D) CD4 + T H cells and CD8 + T C cells. Pooled results from three independent experiments with control n=14 and a2V-KO n=11, mean ± SEM, Mann-Whitney U test, * p
Figure Legend Snippet: a2V deletion in HSCs cause altered recruitment of immune cell populations into the TME After 14 days of implantation, tumors from control and a2V-KO mice were harvested, mechanically disrupted, and digested with Collagenase IV and DNAse I to obtain a single cell suspension. The single cells were enriched for CD45 expression by MACS and subjected to flow-cytometry and counted manually with trypan blue. Representative histograms are shown in the left side. Bar graphs show the number of single, live, CD45 + cells per gram of tumor in the TME. (A) MDSC (B) T cells (C) αβ T cells and γδ T cells (D) CD4 + T H cells and CD8 + T C cells. Pooled results from three independent experiments with control n=14 and a2V-KO n=11, mean ± SEM, Mann-Whitney U test, * p

Techniques Used: Mouse Assay, Expressing, Magnetic Cell Separation, Flow Cytometry, Cytometry, MANN-WHITNEY

17) Product Images from "Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella"

Article Title: Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella

Journal: mBio

doi: 10.1128/mBio.01973-18

Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P
Figure Legend Snippet: Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P

Techniques Used: Crystal Violet Assay

18) Product Images from "Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome"

Article Title: Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E18-06-0353

Hsf1 DNA binding is impeded by nucleosomes, both in vitro and in vivo. (A) Hsf1 and H3 ChIP-seq signal at HSP82 under NHS conditions, normalized to their maximum displayed values. The region used for nucleosome reconstitution and DNase I footprinting is highlighted. (B) DNA corresponding to the HSP82 upstream region depicted in A (spanning –9 to –353 [ATG = +1]) and 32 P-end labeled on the upper strand was either reacted directly with GST-Hsf1 (lanes 4–7) or following its reconstitution into a dinucleosome (lanes 9–12). Reconstitution was achieved using a 1:1 (wt/wt) HeLa histone: DNA ratio and salt dilution, followed by purification over a glycerol gradient (see Supplemental Figure S4). Both naked DNA and chromatin templates were challenged with increasing amounts of recombinant Hsf1 (lanes 3 and 8 are -Hsf1 controls) and then subjected to DNase I digestion. DNA was purified and electrophoresed on an 8% sequencing gel. (C) Scatter plots of Hsf1 ChIP-seq signal as a function of the strength of the HSE for the NHS, 5-min HS, and 120-min HS states. HSE strength was determined by MEME as a p value corresponding to how well the binding site beneath the summit of each ChIP peak matched the consensus HSE motif (Figure 1F). ChIP-seq signals represent the mean of two biological replicates. (D) As in C, but here each p value was divided by the H3 ChIP-seq signal below the summit of the Hsf1 peak under NHS conditions (H3 ChIP-seq data from Qiu et al. [2016] ). Outliers (gray) consist of Hsf1-independent housekeeping genes.
Figure Legend Snippet: Hsf1 DNA binding is impeded by nucleosomes, both in vitro and in vivo. (A) Hsf1 and H3 ChIP-seq signal at HSP82 under NHS conditions, normalized to their maximum displayed values. The region used for nucleosome reconstitution and DNase I footprinting is highlighted. (B) DNA corresponding to the HSP82 upstream region depicted in A (spanning –9 to –353 [ATG = +1]) and 32 P-end labeled on the upper strand was either reacted directly with GST-Hsf1 (lanes 4–7) or following its reconstitution into a dinucleosome (lanes 9–12). Reconstitution was achieved using a 1:1 (wt/wt) HeLa histone: DNA ratio and salt dilution, followed by purification over a glycerol gradient (see Supplemental Figure S4). Both naked DNA and chromatin templates were challenged with increasing amounts of recombinant Hsf1 (lanes 3 and 8 are -Hsf1 controls) and then subjected to DNase I digestion. DNA was purified and electrophoresed on an 8% sequencing gel. (C) Scatter plots of Hsf1 ChIP-seq signal as a function of the strength of the HSE for the NHS, 5-min HS, and 120-min HS states. HSE strength was determined by MEME as a p value corresponding to how well the binding site beneath the summit of each ChIP peak matched the consensus HSE motif (Figure 1F). ChIP-seq signals represent the mean of two biological replicates. (D) As in C, but here each p value was divided by the H3 ChIP-seq signal below the summit of the Hsf1 peak under NHS conditions (H3 ChIP-seq data from Qiu et al. [2016] ). Outliers (gray) consist of Hsf1-independent housekeeping genes.

Techniques Used: Binding Assay, In Vitro, In Vivo, Chromatin Immunoprecipitation, Footprinting, Labeling, Purification, Recombinant, Sequencing

19) Product Images from "Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus"

Article Title: Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02687

Percent detachment of preformed biofilms obtained with 18 ocular  S. haemolyticus  isolates after treatment with NaIO 4 , proteinase K or DNase I.
Figure Legend Snippet: Percent detachment of preformed biofilms obtained with 18 ocular S. haemolyticus isolates after treatment with NaIO 4 , proteinase K or DNase I.

Techniques Used:

The figure shows the orthogonal views of CLSM images.  (A)  Biofilms formed by ATCC 35984 and SHN65 in the presence of proteinase K and DNase I, and  (B)  Detachment of preformed biofilms by adding NaIO 4 , proteinase K, and DNase I. Acquired the z-stacks of each chamber by CLSM with a Leica TCS SP5 confocal scanning system (Leica Microsystem, Mannheim, Germany) 63× oil objective lens.
Figure Legend Snippet: The figure shows the orthogonal views of CLSM images. (A) Biofilms formed by ATCC 35984 and SHN65 in the presence of proteinase K and DNase I, and (B) Detachment of preformed biofilms by adding NaIO 4 , proteinase K, and DNase I. Acquired the z-stacks of each chamber by CLSM with a Leica TCS SP5 confocal scanning system (Leica Microsystem, Mannheim, Germany) 63× oil objective lens.

Techniques Used: Confocal Laser Scanning Microscopy

20) Product Images from "Iron-Regulated Protein HupB of Mycobacterium tuberculosis Positively Regulates Siderophore Biosynthesis and Is Essential for Growth in Macrophages"

Article Title: Iron-Regulated Protein HupB of Mycobacterium tuberculosis Positively Regulates Siderophore Biosynthesis and Is Essential for Growth in Macrophages

Journal: Journal of Bacteriology

doi: 10.1128/JB.01483-13

Identification of the AT-rich HupB box by DNA footprinting. (A and B) Footprint of the [γ- 32 P]ATP-labeled 216-bp reverse strand of the mbtB promoter DNA protected by IdeR and HupB, respectively, from DNase I digestion. Concentrations of the respective
Figure Legend Snippet: Identification of the AT-rich HupB box by DNA footprinting. (A and B) Footprint of the [γ- 32 P]ATP-labeled 216-bp reverse strand of the mbtB promoter DNA protected by IdeR and HupB, respectively, from DNase I digestion. Concentrations of the respective

Techniques Used: DNA Footprinting, Labeling

21) Product Images from "Mechanisms of the Antifungal Action of Marine Metagenome-Derived Peptide, MMGP1, against Candida albicans"

Article Title: Mechanisms of the Antifungal Action of Marine Metagenome-Derived Peptide, MMGP1, against Candida albicans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069316

Effect of proteinase K and DNase I on DNA–MMGP1 complexes. (a) Treatment of DNA–MMGP1 complexes with proteinase K resulted in detection of plasmid DNA band on the agarose gel. C-Control SK+ plasmid DNA; 1-DNA–MMGP1 complex formed at 0.576 µM concentration of peptide; 2-DNA–MMGP1 complex treated with proteinase K (b) DNase I protection assay. The plasmid DNA (100 ng) was preincubated with the varying concentrations peptides such as, C+-no peptide; 1-0.576 µM; 2-0.288 µM; 3- 0.144 μM; 4-0.072 µM; 5-0.036 µM, respectively followed by treatment with DNase I. The DNase treated plasmid DNA was used as negative control (C-).
Figure Legend Snippet: Effect of proteinase K and DNase I on DNA–MMGP1 complexes. (a) Treatment of DNA–MMGP1 complexes with proteinase K resulted in detection of plasmid DNA band on the agarose gel. C-Control SK+ plasmid DNA; 1-DNA–MMGP1 complex formed at 0.576 µM concentration of peptide; 2-DNA–MMGP1 complex treated with proteinase K (b) DNase I protection assay. The plasmid DNA (100 ng) was preincubated with the varying concentrations peptides such as, C+-no peptide; 1-0.576 µM; 2-0.288 µM; 3- 0.144 μM; 4-0.072 µM; 5-0.036 µM, respectively followed by treatment with DNase I. The DNase treated plasmid DNA was used as negative control (C-).

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Concentration Assay, Negative Control

22) Product Images from "Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance"

Article Title: Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01311-20

CG viewer representation of the PMK34 genome. The outer ring represents the 50 predicted ORFs (arrows) of the phage, with different colors representing the different modular functional groups. The middle and inner rings illustrate the GC skew and GC content, respectively. The PMK34 genome reveals a typical modular organization with DNA replication/transcription, morphogenesis, host lysis, and DNA packaging genes, apart from a region dominated by hypothetical genes. The DNA replication/transcription gene group (11 ORFs) comprises genes encoding the DNA primase/helicase ( orf17  and  orf21 ), DNA ligase ( orf23 ), DNA polymerase I ( orf25 ), DNA exonuclease ( orf28 ), tRNA nucleotidyltransferase ( orf29 ), DNA endonuclease VII ( orf30 ), phosphoesterase ( orf31 ), dNMP kinase ( orf32 ), and single subunit RNA polymerase ( orf33 ). Only a single HNH endonuclease-encoding gene ( orf24 ) was found upstream of the DNA polymerase I ( orf25 ) sequence. The DNA replication/transcription gene group is followed by morphogenesis gene group separated by two hypothetical proteins ( orf34  and  orf35 ). Different structural proteins encoded by this gene cluster include the head tail connector ( orf36 ), scaffold protein ( orf37 ), capsid ( orf38 ), tail tubular proteins A ( orf40 ) and B ( orf41 ), internal virion proteins ( orf42  and  orf44 ), and a tail fiber with putative pectate lyase domain ( orf45 ). The host cell lysis gene group comprises two overlapping genes, i.e., the holin gene ( orf46 ) located upstream of the endolysin sequence ( orf47 ). No spanin gene is detected. The holin protein has three predicted transmembrane helices with N-out and C-in topology, being a member of the class I holin. The final predicted proteins are the DNA packaging and maturase genes A ( orf48 ) and B ( orf49 ).
Figure Legend Snippet: CG viewer representation of the PMK34 genome. The outer ring represents the 50 predicted ORFs (arrows) of the phage, with different colors representing the different modular functional groups. The middle and inner rings illustrate the GC skew and GC content, respectively. The PMK34 genome reveals a typical modular organization with DNA replication/transcription, morphogenesis, host lysis, and DNA packaging genes, apart from a region dominated by hypothetical genes. The DNA replication/transcription gene group (11 ORFs) comprises genes encoding the DNA primase/helicase ( orf17 and orf21 ), DNA ligase ( orf23 ), DNA polymerase I ( orf25 ), DNA exonuclease ( orf28 ), tRNA nucleotidyltransferase ( orf29 ), DNA endonuclease VII ( orf30 ), phosphoesterase ( orf31 ), dNMP kinase ( orf32 ), and single subunit RNA polymerase ( orf33 ). Only a single HNH endonuclease-encoding gene ( orf24 ) was found upstream of the DNA polymerase I ( orf25 ) sequence. The DNA replication/transcription gene group is followed by morphogenesis gene group separated by two hypothetical proteins ( orf34 and orf35 ). Different structural proteins encoded by this gene cluster include the head tail connector ( orf36 ), scaffold protein ( orf37 ), capsid ( orf38 ), tail tubular proteins A ( orf40 ) and B ( orf41 ), internal virion proteins ( orf42 and orf44 ), and a tail fiber with putative pectate lyase domain ( orf45 ). The host cell lysis gene group comprises two overlapping genes, i.e., the holin gene ( orf46 ) located upstream of the endolysin sequence ( orf47 ). No spanin gene is detected. The holin protein has three predicted transmembrane helices with N-out and C-in topology, being a member of the class I holin. The final predicted proteins are the DNA packaging and maturase genes A ( orf48 ) and B ( orf49 ).

Techniques Used: Functional Assay, Lysis, Sequencing

23) Product Images from "RORα enforces stability of the T-helper-17 cell effector program"

Article Title: RORα enforces stability of the T-helper-17 cell effector program

Journal: bioRxiv

doi: 10.1101/2020.12.15.422921

Efficient editing of  Rorc(t)  +11kb  cis -element by CAS9-RNP method. Related to   Figure 6 . (A)  Analysis of CAS9/gRNA RNP-mediated targeting efficiency of +11kb enhancer by T7 endonuclease I assay. (B)  Sanger sequencing results displaying  Rorc(t)  +11kb enhancer mutations and deletions of T AKO  +11kb ΔRORE  2D2tg-Th17 cells. (C)  Experimental scheme to examine the role of  Rorc(t)  +11kb  cis -element in maintenance of pathogenic Th17 program during EAE.
Figure Legend Snippet: Efficient editing of Rorc(t) +11kb cis -element by CAS9-RNP method. Related to Figure 6 . (A) Analysis of CAS9/gRNA RNP-mediated targeting efficiency of +11kb enhancer by T7 endonuclease I assay. (B) Sanger sequencing results displaying Rorc(t) +11kb enhancer mutations and deletions of T AKO +11kb ΔRORE 2D2tg-Th17 cells. (C) Experimental scheme to examine the role of Rorc(t) +11kb cis -element in maintenance of pathogenic Th17 program during EAE.

Techniques Used: T7EI Assay, Sequencing

24) Product Images from "STING-dependent sensing of self-DNA drives silica-induced lung inflammation"

Article Title: STING-dependent sensing of self-DNA drives silica-induced lung inflammation

Journal: Nature Communications

doi: 10.1038/s41467-018-07425-1

Extracellular self-dsDNA is key to silica-induced inflammatory response in macrophages, not in DCs. a – c Extracellular DNase I treatment (1 µg/mL) was applied 3 h prior and 1 h after silica exposure (250 µg/mL) or transfection with c-di-AMP (6 µg/mL; cDN) for 18 h in bone marrow-derived macrophages ( b ) and dentritic cells ( c ). b Macrophage concentration of extracellular dsDNA and CXCL10 in culture supernatant. Ifnα and Ifnβ transcripts measured by real-time PCR on cell fractions and IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. c Dendritic cell IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. d – i Mitochondrial DNA replication was inhibited using a low concentration of EtdBr (150 ng/mL) on day 7 of bone marrow cell culture. On day 11, bone marrow-derived DCs ( e – h ) and macrophages ( i ) were unstimulated, stimulated with silica (250 µg/mL), or transfected with c-di-AMP (6 µg/mL; cDN) for 18 h. Fold change of ( e ) mitochondrial DNA (mMitoF1 and mMitoR1) and ( f ) nuclear DNA (mB2MF1 and mB2MR1) in untreated versus EtdBr-treated DC exposed to silica, as compared to untreated unstimulated cells. g Immunoblot showing cGAS, phospho-STING, STING protein expression, and dimerization in DCs after EtdBr treatment, with β-actin as a reference. h CXCL10 level in DC supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. i CXCL10 level in macrophage supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. * p
Figure Legend Snippet: Extracellular self-dsDNA is key to silica-induced inflammatory response in macrophages, not in DCs. a – c Extracellular DNase I treatment (1 µg/mL) was applied 3 h prior and 1 h after silica exposure (250 µg/mL) or transfection with c-di-AMP (6 µg/mL; cDN) for 18 h in bone marrow-derived macrophages ( b ) and dentritic cells ( c ). b Macrophage concentration of extracellular dsDNA and CXCL10 in culture supernatant. Ifnα and Ifnβ transcripts measured by real-time PCR on cell fractions and IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. c Dendritic cell IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. d – i Mitochondrial DNA replication was inhibited using a low concentration of EtdBr (150 ng/mL) on day 7 of bone marrow cell culture. On day 11, bone marrow-derived DCs ( e – h ) and macrophages ( i ) were unstimulated, stimulated with silica (250 µg/mL), or transfected with c-di-AMP (6 µg/mL; cDN) for 18 h. Fold change of ( e ) mitochondrial DNA (mMitoF1 and mMitoR1) and ( f ) nuclear DNA (mB2MF1 and mB2MR1) in untreated versus EtdBr-treated DC exposed to silica, as compared to untreated unstimulated cells. g Immunoblot showing cGAS, phospho-STING, STING protein expression, and dimerization in DCs after EtdBr treatment, with β-actin as a reference. h CXCL10 level in DC supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. i CXCL10 level in macrophage supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. * p

Techniques Used: Transfection, Derivative Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Multiplex Assay, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Self-dsDNA release is central to silica-induced lung inflammation and type I IFN response. a – m Silica microparticles (1 mg/mouse, i.t.) or saline were administered in WT mice and parameters were analyzed on day 7. a Concentration of extracellular dsDNA in the acellular fractions of bronchoalveolar lavage fluid (BALF). b – d Tmem173 , Mb21d1, Ifnα, and Ifnβ transcripts in the lungs and normalized to Gapdh expression. e Lung CXCL10 determined by ELISA and IFN-αβ proteins quantified by multiplex immunoassay. f Correlation between extracellular dsDNA concentrations and type I IFN gene expression. g Annexin V/PI flow cytometry analysis pre-gated on singlet cells. h Correlation between dead cells and extracellular dsDNA. i Increased nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in the BALF after silica exposure. j Immunoblots of caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), gasdermin D (GSDMD), MLKL, and phosphorylated-MLKL (p-MLKL), normalized to β-actin. Immunoblot quantifications of ( k ) c-Casp3, ( l ) c-GSDMD, and ( m ) p-MLKL. n – u Silica microparticles administered with DNase I (200 µg/mouse, i.p.) as indicated ( n ). o Extracellular dsDNA in BALF acellular fraction. p Lung immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), MLKL, and phosphorylated-MLKL (p-MLKL), with β-actin as a reference. q Quantification of STING dimer, relative to β-actin. r STING immunoblots under 5% reducing (left) or 1% 2-mercaptoethanol seminative conditions (2-ME; right). s Lung confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 20 µm. t Pulmonary IFN-αβ and CXCL10. u Neutrophils, macrophages, and protein extravasation in the BALF. * p
Figure Legend Snippet: Self-dsDNA release is central to silica-induced lung inflammation and type I IFN response. a – m Silica microparticles (1 mg/mouse, i.t.) or saline were administered in WT mice and parameters were analyzed on day 7. a Concentration of extracellular dsDNA in the acellular fractions of bronchoalveolar lavage fluid (BALF). b – d Tmem173 , Mb21d1, Ifnα, and Ifnβ transcripts in the lungs and normalized to Gapdh expression. e Lung CXCL10 determined by ELISA and IFN-αβ proteins quantified by multiplex immunoassay. f Correlation between extracellular dsDNA concentrations and type I IFN gene expression. g Annexin V/PI flow cytometry analysis pre-gated on singlet cells. h Correlation between dead cells and extracellular dsDNA. i Increased nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in the BALF after silica exposure. j Immunoblots of caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), gasdermin D (GSDMD), MLKL, and phosphorylated-MLKL (p-MLKL), normalized to β-actin. Immunoblot quantifications of ( k ) c-Casp3, ( l ) c-GSDMD, and ( m ) p-MLKL. n – u Silica microparticles administered with DNase I (200 µg/mouse, i.p.) as indicated ( n ). o Extracellular dsDNA in BALF acellular fraction. p Lung immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), MLKL, and phosphorylated-MLKL (p-MLKL), with β-actin as a reference. q Quantification of STING dimer, relative to β-actin. r STING immunoblots under 5% reducing (left) or 1% 2-mercaptoethanol seminative conditions (2-ME; right). s Lung confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 20 µm. t Pulmonary IFN-αβ and CXCL10. u Neutrophils, macrophages, and protein extravasation in the BALF. * p

Techniques Used: Mouse Assay, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Flow Cytometry, Cytometry, Western Blot

25) Product Images from "Neutrophil extracellular traps promote scar formation in post-epidural fibrosis"

Article Title: Neutrophil extracellular traps promote scar formation in post-epidural fibrosis

Journal: NPJ Regenerative Medicine

doi: 10.1038/s41536-020-00103-1

DNase I alleviated epidural fibrosis in the mouse model of laminectomy. A Gross observation demonstrated that DNase I reduced scar formation in the operation group; B MRI confirmed that there was a gap between the scar tissue and the dura mater after DNase treatment, indicating that the degree of scar adhesion decreased; C immunofluorescence staining attested that DNase reduced the generation of NETs (citH3, pseudo-red; MPO, pseudo-green) in the wound area after spine operation; D H E staining showed the histological morphology of each group of scars. The values within the table represented the number of mice. E Masson staining showed the accumulation of collagen fibers in each group of scars; F DNase I reduced collagen I in the scar tissue from the operation group. Error bars = SEM; Student’s t test for two groups comparison, two-way ANOVA for three and more groups comparison, number indicated in the figure; * p
Figure Legend Snippet: DNase I alleviated epidural fibrosis in the mouse model of laminectomy. A Gross observation demonstrated that DNase I reduced scar formation in the operation group; B MRI confirmed that there was a gap between the scar tissue and the dura mater after DNase treatment, indicating that the degree of scar adhesion decreased; C immunofluorescence staining attested that DNase reduced the generation of NETs (citH3, pseudo-red; MPO, pseudo-green) in the wound area after spine operation; D H E staining showed the histological morphology of each group of scars. The values within the table represented the number of mice. E Masson staining showed the accumulation of collagen fibers in each group of scars; F DNase I reduced collagen I in the scar tissue from the operation group. Error bars = SEM; Student’s t test for two groups comparison, two-way ANOVA for three and more groups comparison, number indicated in the figure; * p

Techniques Used: Magnetic Resonance Imaging, Immunofluorescence, Staining, Mouse Assay

NETs promoted the macrophage phenotype transition. A NETs increased the expression of α-SMA and fibronectin in macrophages. B In vitro immunofluorescence analysis revealed that NETs-treated macrophages coexpressed F4/80 and α-SMA. C In the spinal tissues 2 days post operation, cells co-expressing F4/80 and α-SMA were observed. Similarly, cells coexpressed F4/80 and collagen I were also recorded. D DNase I reduced the expression of fibronectin in NETs-treated macrophages. E The RAGE inhibitor FPS-ZM1 reduced fibronectin expression in NETs-treated macrophages. All blots or gels derive from the same experiment and that they were processed in parallel. Error bars = SEM; Student’s t test for two groups comparison, two-way ANOVA for three and more groups comparison, number indicated in the figure; * p
Figure Legend Snippet: NETs promoted the macrophage phenotype transition. A NETs increased the expression of α-SMA and fibronectin in macrophages. B In vitro immunofluorescence analysis revealed that NETs-treated macrophages coexpressed F4/80 and α-SMA. C In the spinal tissues 2 days post operation, cells co-expressing F4/80 and α-SMA were observed. Similarly, cells coexpressed F4/80 and collagen I were also recorded. D DNase I reduced the expression of fibronectin in NETs-treated macrophages. E The RAGE inhibitor FPS-ZM1 reduced fibronectin expression in NETs-treated macrophages. All blots or gels derive from the same experiment and that they were processed in parallel. Error bars = SEM; Student’s t test for two groups comparison, two-way ANOVA for three and more groups comparison, number indicated in the figure; * p

Techniques Used: Expressing, In Vitro, Immunofluorescence

26) Product Images from "Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells"

Article Title: Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1181-x

Activation of DNA damage and inflammation in brain cells of mice delivered lower hemi-body and focused mini-beam radiations and their abrogation by concurrent treatment with cfCh inactivating agents. A Dose-response effect of out-of-field RIBE following lower HBI in brain cells with respect to γH2AX and NFκB. B Graph showing surge of cfCh in circulation at 36 h following lower HBI measured by Cell Death Detection ELISA plus kit wherein results are expressed in terms of absorbance kinetics at 405 nm. C Inhibition of out-of-field RIBE with respect to γH2AX, active Caspase -3, NFκB, and IL-6 by CNPs, DNase I and R-Cu. All animals except the control group received lower HBI (10 Gy) with and without CNPs, DNase I, and R-Cu. D Inhibition of out-of-field RIBE with respect to γH2AX and NFκB. All animals except the control group received focused min-beam radiation (20 Gy) with and without CNPs, DNase I, and R-Cu. The control and irradiated groups comprising five animals each while those receiving radiation plus CNPs, DNase I and R-Cu comprised of three animals each. All histograms depict mean MFI (±S.E.). The groups were compared using Student’s t -test. * p
Figure Legend Snippet: Activation of DNA damage and inflammation in brain cells of mice delivered lower hemi-body and focused mini-beam radiations and their abrogation by concurrent treatment with cfCh inactivating agents. A Dose-response effect of out-of-field RIBE following lower HBI in brain cells with respect to γH2AX and NFκB. B Graph showing surge of cfCh in circulation at 36 h following lower HBI measured by Cell Death Detection ELISA plus kit wherein results are expressed in terms of absorbance kinetics at 405 nm. C Inhibition of out-of-field RIBE with respect to γH2AX, active Caspase -3, NFκB, and IL-6 by CNPs, DNase I and R-Cu. All animals except the control group received lower HBI (10 Gy) with and without CNPs, DNase I, and R-Cu. D Inhibition of out-of-field RIBE with respect to γH2AX and NFκB. All animals except the control group received focused min-beam radiation (20 Gy) with and without CNPs, DNase I, and R-Cu. The control and irradiated groups comprising five animals each while those receiving radiation plus CNPs, DNase I and R-Cu comprised of three animals each. All histograms depict mean MFI (±S.E.). The groups were compared using Student’s t -test. * p

Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Irradiation

Filtered irradiated conditioned medium induce DNA damage, apoptosis, and inflammation in bystander cells. Various donor cells were irradiated (10 Gy) and after 6 h of incubation the culture media were passed through 0.22 µm filters. The filtered media were incubated with recipient cells for 6 h and analyzed for activation of RIBE. A – D The experiments were done in four different combinations of donor and recipient cell lines as depicted in the figure. Conditioned media from irradiated cells markedly activated H2AX, active Caspase-3, NFκB, and IL-6 in all cellular combinations. Pre-treatment of irradiated filtered conditioned medium with CNPs, DNase I and R-Cu completely prevented RIBE. The experiments were done in duplicate and the histograms depict mean MFI (±S.E.). The groups were compared using Student’s t -test. * p
Figure Legend Snippet: Filtered irradiated conditioned medium induce DNA damage, apoptosis, and inflammation in bystander cells. Various donor cells were irradiated (10 Gy) and after 6 h of incubation the culture media were passed through 0.22 µm filters. The filtered media were incubated with recipient cells for 6 h and analyzed for activation of RIBE. A – D The experiments were done in four different combinations of donor and recipient cell lines as depicted in the figure. Conditioned media from irradiated cells markedly activated H2AX, active Caspase-3, NFκB, and IL-6 in all cellular combinations. Pre-treatment of irradiated filtered conditioned medium with CNPs, DNase I and R-Cu completely prevented RIBE. The experiments were done in duplicate and the histograms depict mean MFI (±S.E.). The groups were compared using Student’s t -test. * p

Techniques Used: Irradiation, Incubation, Activation Assay

27) Product Images from "Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism"

Article Title: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism

Journal: Structure (London, England : 1993)

doi: 10.1016/j.str.2009.05.012

DNase I footprinting and in vitro transcription assay
Figure Legend Snippet: DNase I footprinting and in vitro transcription assay

Techniques Used: Footprinting, In Vitro

28) Product Images from "CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp"

Article Title: CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001048

Identification of a CTCF binding site upstream of Qp. A) Schematic of CTCF and EBNA1 binding site organization at Qp, and the sequence of candidate CTCF binding sties (BS) 1 and 2. B) Coomassie stain of purified recombinant CTCF and EBNA1 proteins derived from baculovirus expression system. C) EMSA analysis of purified CTCF (10–100 ng) binding to DNA probes for EBV regions 49730–49768 (BS1), 49901–49939, or 50072–50113 (BS2). D) DNase I footprinting assay of purified CTCF protein (30–300 ng) in the absence (-) or addition (+) of 30 ng purified EBNA1, in buffer containing 150 mM (left panel) or 75 mM (right panel) NaCl.
Figure Legend Snippet: Identification of a CTCF binding site upstream of Qp. A) Schematic of CTCF and EBNA1 binding site organization at Qp, and the sequence of candidate CTCF binding sties (BS) 1 and 2. B) Coomassie stain of purified recombinant CTCF and EBNA1 proteins derived from baculovirus expression system. C) EMSA analysis of purified CTCF (10–100 ng) binding to DNA probes for EBV regions 49730–49768 (BS1), 49901–49939, or 50072–50113 (BS2). D) DNase I footprinting assay of purified CTCF protein (30–300 ng) in the absence (-) or addition (+) of 30 ng purified EBNA1, in buffer containing 150 mM (left panel) or 75 mM (right panel) NaCl.

Techniques Used: Binding Assay, Sequencing, Staining, Purification, Recombinant, Derivative Assay, Expressing, Footprinting

29) Product Images from "Neutrophil extracellular trap stabilization leads to improved outcomes in murine models of sepsis"

Article Title: Neutrophil extracellular trap stabilization leads to improved outcomes in murine models of sepsis

Journal: bioRxiv

doi: 10.1101/630483

Effect of PF4 on endothelial cells and microbial entrapment by NETs in vitro. Channels lined with TNFα-stimulated HUVECs were infused with isolated human neutrophils treated with TNFα (1 ng/ml) with and without hPF4 (25 μg/ml). ( A ) Channels were incubated with the neutrophils for 16 hours after which the number of residual adherent endothelial cells was counted. At the top, representative images of remaining attached endothelial cells channels per condition. Size-bar and arrows indicating direction of flow are included. At the bottom, mean of endothelial cell counts in three 10x high-powered fields (HPFs) per condition ± 1 SD. Statistical analysis was performed using a Student’s test. ( B ) Left shows representative images of NET-lined channels infused with fluorescently-labeled S. aureus with observed bacterial capture. Size-bar and arrows indicating direction of flow are included. Right shows mean ± 1 SD quantified of the mean fluorescent intensity (MFI). For both left and right, top are studies without added DNase I and bottom in the presence of DNase I (100U/ml). N = 5-10 channels per condition. Analysis performed by a Kruskal-Wallis one-way ANOVA.
Figure Legend Snippet: Effect of PF4 on endothelial cells and microbial entrapment by NETs in vitro. Channels lined with TNFα-stimulated HUVECs were infused with isolated human neutrophils treated with TNFα (1 ng/ml) with and without hPF4 (25 μg/ml). ( A ) Channels were incubated with the neutrophils for 16 hours after which the number of residual adherent endothelial cells was counted. At the top, representative images of remaining attached endothelial cells channels per condition. Size-bar and arrows indicating direction of flow are included. At the bottom, mean of endothelial cell counts in three 10x high-powered fields (HPFs) per condition ± 1 SD. Statistical analysis was performed using a Student’s test. ( B ) Left shows representative images of NET-lined channels infused with fluorescently-labeled S. aureus with observed bacterial capture. Size-bar and arrows indicating direction of flow are included. Right shows mean ± 1 SD quantified of the mean fluorescent intensity (MFI). For both left and right, top are studies without added DNase I and bottom in the presence of DNase I (100U/ml). N = 5-10 channels per condition. Analysis performed by a Kruskal-Wallis one-way ANOVA.

Techniques Used: In Vitro, Isolation, Incubation, Labeling

Schematic of PF4- and KKO-mediated NET compaction and NDP sequestration. Neutrophils release NETs after the enzyme peptidyl arginine deiminase 4 (PAD4) citrullinates histones (His) to become cit-His. Toxic NET degradation products (NDPs) such as cfDNA and histones, are released from NETs after DNase I digestion, leading to platelet activation and endothelial damage. At the same time, activated platelets release PF4 that binds to NETs, causing them to become dense, compact and partially resistant to DNase I. It also enhances their ability to capture bacteria. DG-KKO binds to PF4-NET complexes, enhancing their resistance to DNase I, sequestrating NDPs, and improving outcome.
Figure Legend Snippet: Schematic of PF4- and KKO-mediated NET compaction and NDP sequestration. Neutrophils release NETs after the enzyme peptidyl arginine deiminase 4 (PAD4) citrullinates histones (His) to become cit-His. Toxic NET degradation products (NDPs) such as cfDNA and histones, are released from NETs after DNase I digestion, leading to platelet activation and endothelial damage. At the same time, activated platelets release PF4 that binds to NETs, causing them to become dense, compact and partially resistant to DNase I. It also enhances their ability to capture bacteria. DG-KKO binds to PF4-NET complexes, enhancing their resistance to DNase I, sequestrating NDPs, and improving outcome.

Techniques Used: Activation Assay

Binding of DG-KKO to PF4/NET complexes in vitro. ( A ) Graphs quantifying binding of increasing concentrations of KKO (gray) and DG-KKO (red) to heparin using fluorescent plate assay. ( B ) same as (A) but using flow cytometry to quantify antibody binding to the platelet surface. ( C ) Mean ± 1 SD of P-selectin MFI in human whole blood samples incubated with the indicated concentration of antibody, reflecting the degree of platelet activation. N=3. ( D ) Mean ± 1 SD of the % decrease in platelets counts in HIT mice injected with 400μg of the indicated antibody, measured every 12 hours for 3 days. N=3. ( E ) Representative confocal images of released NETs as in Figure 2 exposed to no PF4 or 6.5 μg/ml of PF4, labeled with the nucleic acid stain Sytox green (green) demonstrating change in morphology. The indicated channels were then infused with fluorophore-labeled DG-KKO (white). Size-bar and arrows indicating direction of flow are included. Image were obtained at 10x magnification. ( F ) Representative widefield images of adherent neutrophils as in (A), but in the presence of 100U/ml DNase I and 6.5 μg/ml PF4 ± 25 μg/ml of DG-KKO. ( G ) Mean ± 1 SD of the relative area of NETs compacted with PF4 (6.5 μg/ml) alone or PF4 plus either DG-KKP or a polyclonal anti-PF4 antibody control (Ctl) (each, 25 μg/ml KKO) post an infusion of DNase I (100U/ml, 3 minutes) compared to pre-infusion. N = 7-10 channels per condition. Comparative statistical analysis performed by Kruskall-Wallis one-way ANOVA.
Figure Legend Snippet: Binding of DG-KKO to PF4/NET complexes in vitro. ( A ) Graphs quantifying binding of increasing concentrations of KKO (gray) and DG-KKO (red) to heparin using fluorescent plate assay. ( B ) same as (A) but using flow cytometry to quantify antibody binding to the platelet surface. ( C ) Mean ± 1 SD of P-selectin MFI in human whole blood samples incubated with the indicated concentration of antibody, reflecting the degree of platelet activation. N=3. ( D ) Mean ± 1 SD of the % decrease in platelets counts in HIT mice injected with 400μg of the indicated antibody, measured every 12 hours for 3 days. N=3. ( E ) Representative confocal images of released NETs as in Figure 2 exposed to no PF4 or 6.5 μg/ml of PF4, labeled with the nucleic acid stain Sytox green (green) demonstrating change in morphology. The indicated channels were then infused with fluorophore-labeled DG-KKO (white). Size-bar and arrows indicating direction of flow are included. Image were obtained at 10x magnification. ( F ) Representative widefield images of adherent neutrophils as in (A), but in the presence of 100U/ml DNase I and 6.5 μg/ml PF4 ± 25 μg/ml of DG-KKO. ( G ) Mean ± 1 SD of the relative area of NETs compacted with PF4 (6.5 μg/ml) alone or PF4 plus either DG-KKP or a polyclonal anti-PF4 antibody control (Ctl) (each, 25 μg/ml KKO) post an infusion of DNase I (100U/ml, 3 minutes) compared to pre-infusion. N = 7-10 channels per condition. Comparative statistical analysis performed by Kruskall-Wallis one-way ANOVA.

Techniques Used: Binding Assay, In Vitro, Flow Cytometry, Incubation, Concentration Assay, Activation Assay, Mouse Assay, Injection, Labeling, Staining

30) Product Images from "MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis"

Article Title: MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-017-0915-5

MHC class II expression correlates to EAU severity. Three weeks after adoptive transfer, retinas were carefully dissected, cut into small pieces, and dissociated by incubation with Liberase DL and DNase I at 37 °C for 45 min. Naive eyes were used as control. The single-cell suspensions, excluding dead cells (DAPI+) were analyzed by flow cytometry for MHC II expression using fluorochrome-conjugated specific antibodies. For Figures B to E, data are from 1 representative mouse out of 21 independent mice (5 control, 8 low-score EAU and 8 high-score EAU mice). a Number of MHC class II-expressing cells (normalized per 1 million analyzed retinal cells) for different EAU clinical scores. Data represented: mean ± SEM, ANOVA, and Tukey post-hoc multiple comparisons test, ** p
Figure Legend Snippet: MHC class II expression correlates to EAU severity. Three weeks after adoptive transfer, retinas were carefully dissected, cut into small pieces, and dissociated by incubation with Liberase DL and DNase I at 37 °C for 45 min. Naive eyes were used as control. The single-cell suspensions, excluding dead cells (DAPI+) were analyzed by flow cytometry for MHC II expression using fluorochrome-conjugated specific antibodies. For Figures B to E, data are from 1 representative mouse out of 21 independent mice (5 control, 8 low-score EAU and 8 high-score EAU mice). a Number of MHC class II-expressing cells (normalized per 1 million analyzed retinal cells) for different EAU clinical scores. Data represented: mean ± SEM, ANOVA, and Tukey post-hoc multiple comparisons test, ** p

Techniques Used: Expressing, Adoptive Transfer Assay, Incubation, Flow Cytometry, Mouse Assay

31) Product Images from "Clp ATPases differentially affect natural competence development in Streptococcus mutans). Clp ATPases differentially affect natural competence development in Streptococcus mutans"

Article Title: Clp ATPases differentially affect natural competence development in Streptococcus mutans). Clp ATPases differentially affect natural competence development in Streptococcus mutans

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.1288

Experimental strategy to evaluate transformation efficiency in UA159. (a) Schematic representation of the strategy to evaluate DNA uptake using DNase I (+). At culture OD 600 = 0.15 (considered time zero; t , 0), DNA (pIB184Em; 1 µg/ml) and CSP18 (200 nM) were added, and the culture was equally distributed (5 ml each) in four groups (A–D). In Group A, DNase I (10 U) was added immediately ( t , 0). In Group B, DNase I was added 15 min before plating (P) at 1 h followed by plating at different time points. In Group C, culture was further distributed equally in five vials (1 ml each), and DNase I was added into each vial at different time points just 15 min before plating. In Group D, no DNase I was added. Plating of an aliquot from the four groups was done at 1, 1.5, 2, 2.5, and 3 h of growth at 37°C. (b) Transformation efficiency plot of four different groups (a–d). The y axis is represented on the log10 scale and Group A was not shown in the graph due to the “zero” value. The values represent the “mean ± SD“ of three independent replicates. Statistical significance (*) between Groups C and D was calculated using a t‐ test.
Figure Legend Snippet: Experimental strategy to evaluate transformation efficiency in UA159. (a) Schematic representation of the strategy to evaluate DNA uptake using DNase I (+). At culture OD 600 = 0.15 (considered time zero; t , 0), DNA (pIB184Em; 1 µg/ml) and CSP18 (200 nM) were added, and the culture was equally distributed (5 ml each) in four groups (A–D). In Group A, DNase I (10 U) was added immediately ( t , 0). In Group B, DNase I was added 15 min before plating (P) at 1 h followed by plating at different time points. In Group C, culture was further distributed equally in five vials (1 ml each), and DNase I was added into each vial at different time points just 15 min before plating. In Group D, no DNase I was added. Plating of an aliquot from the four groups was done at 1, 1.5, 2, 2.5, and 3 h of growth at 37°C. (b) Transformation efficiency plot of four different groups (a–d). The y axis is represented on the log10 scale and Group A was not shown in the graph due to the “zero” value. The values represent the “mean ± SD“ of three independent replicates. Statistical significance (*) between Groups C and D was calculated using a t‐ test.

Techniques Used: Transformation Assay

32) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1191

The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.
Figure Legend Snippet: The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

Techniques Used: Plasmid Preparation, Incubation, Activity Assay

gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
Figure Legend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

Techniques Used: Incubation, Plasmid Preparation, Labeling

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    Millipore dnase i
    The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. <t>DNase</t> I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore deoxyribonuclease i from bovine pancreas
    Contact-enhanced activation of DCs by DHMs. BMDCs were incubated with DHMs or nmDHMs for 18 hours, after which DHMs/nmDHMs were removed from the well and digested with DNase I. Cells attached to and surrounding DHMs were analyzed for surface marker expression of CD40, CD80, and CD86 by flow cytometry. ** p
    Deoxyribonuclease I From Bovine Pancreas, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxyribonuclease i from bovine pancreas/product/Millipore
    Average 99 stars, based on 1 article reviews
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    deoxyribonuclease i from bovine pancreas - by Bioz Stars, 2022-08
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    The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. DNase I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.

    Journal: PLoS ONE

    Article Title: Obesity Risk Gene TMEM18 Encodes a Sequence-Specific DNA-Binding Protein

    doi: 10.1371/journal.pone.0025317

    Figure Lengend Snippet: The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C's indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. DNase I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays.

    Article Snippet: Dnase I treatment of the matrix was done as follows: the matrix was washed three times with 10 mM Tris pH 7.5, 0.2 M NaCl and incubated with 10 mg/ml of Dnase I (Sigma) in 10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2 for 15 minutes at room temperature (RT).

    Techniques: Binding Assay, Western Blot, Flow Cytometry

    Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p

    Journal: Cell Death & Disease

    Article Title: The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

    doi: 10.1038/s41419-018-0721-8

    Figure Lengend Snippet: Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p

    Article Snippet: Two equal aliquots of 75 µl of nuclei were treated with 0 or 2 units of DNase I (Sigma, USA) at 37 °C for 5 min.

    Techniques: Expressing, Isolation, Mouse Assay, Transplantation Assay, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Luciferase

    To compare Benzonase to DNase I: SH-SY5Y cells were digested with various concentrations of Benzonase (A) or DNase I (B). qPCR indicated that Benzonase digestion exhibited similar patterns of relative openness to DNase I digestion in 7 distinct regions

    Journal: Neuroepigenetics

    Article Title: Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells

    doi: 10.1016/j.nepig.2016.03.001

    Figure Lengend Snippet: To compare Benzonase to DNase I: SH-SY5Y cells were digested with various concentrations of Benzonase (A) or DNase I (B). qPCR indicated that Benzonase digestion exhibited similar patterns of relative openness to DNase I digestion in 7 distinct regions

    Article Snippet: The nuclei were aliquoted equally into 5 microfuge tubes containing different concentrations of DNase I (Sigma) (None, 0.12U, 0.4U, 1.2U, 4U).

    Techniques: Real-time Polymerase Chain Reaction

    Contact-enhanced activation of DCs by DHMs. BMDCs were incubated with DHMs or nmDHMs for 18 hours, after which DHMs/nmDHMs were removed from the well and digested with DNase I. Cells attached to and surrounding DHMs were analyzed for surface marker expression of CD40, CD80, and CD86 by flow cytometry. ** p

    Journal: Advanced healthcare materials

    Article Title: Extracellular trap-mimicking DNA-histone mesostructures synergistically activate dendritic cells

    doi: 10.1002/adhm.201900926

    Figure Lengend Snippet: Contact-enhanced activation of DCs by DHMs. BMDCs were incubated with DHMs or nmDHMs for 18 hours, after which DHMs/nmDHMs were removed from the well and digested with DNase I. Cells attached to and surrounding DHMs were analyzed for surface marker expression of CD40, CD80, and CD86 by flow cytometry. ** p

    Article Snippet: For each replicate, the 36 DHMs were forcefully pipetted into a separate container and centrifuged, after which the supernatant was removed and replaced with 10 U/mL DNase I (Item No D5025, Millipore Sigma) in a Ca2+ and Mg2+-supplemented 10 mM Tris-HCl buffer.

    Techniques: Activation Assay, Incubation, Marker, Expressing, Flow Cytometry