gfp negative facs  (Qiagen)


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    DNeasy Blood Tissue Kit
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    For spin column or 96 well purification of total DNA from animal blood and tissues and from cells yeast bacteria or viruses Kit contents Qiagen DNeasy Blood Tissue Kit 50 preps 100L 25mg Sample 100 to 200L Elution Volume Blood Tissue Sample Total DNA Purification Silica Technology Spin Column Format 6g 30g Yield Manual Processing For Purification of Total DNA from Animal Blood and Tissues and from Cells Yeast Bacteria or Viruses Ideal for PCR Real time PCR Genotyping Includes 50 DNeasy Mini Spin Columns Proteinase K Buffers 2mL Collection Tubes Benefits Standardized method for a variety of sample types High yields even from specialized samples High quality DNA Optimized protocols for a range of starting materials Spin column and 96 well high throughput form
    Catalog Number:
    69504
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    DNeasy Blood Tissue Kits
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    Structured Review

    Qiagen gfp negative facs
    DNeasy Blood Tissue Kit
    For spin column or 96 well purification of total DNA from animal blood and tissues and from cells yeast bacteria or viruses Kit contents Qiagen DNeasy Blood Tissue Kit 50 preps 100L 25mg Sample 100 to 200L Elution Volume Blood Tissue Sample Total DNA Purification Silica Technology Spin Column Format 6g 30g Yield Manual Processing For Purification of Total DNA from Animal Blood and Tissues and from Cells Yeast Bacteria or Viruses Ideal for PCR Real time PCR Genotyping Includes 50 DNeasy Mini Spin Columns Proteinase K Buffers 2mL Collection Tubes Benefits Standardized method for a variety of sample types High yields even from specialized samples High quality DNA Optimized protocols for a range of starting materials Spin column and 96 well high throughput form
    https://www.bioz.com/result/gfp negative facs/product/Qiagen
    Average 93 stars, based on 14948 article reviews
    Price from $9.99 to $1999.99
    gfp negative facs - by Bioz Stars, 2020-04
    93/100 stars

    Images

    1) Product Images from "Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells"

    Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells

    Journal: eLife

    doi: 10.7554/eLife.36187

    GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.
    Figure Legend Snippet: GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.

    Techniques Used: FACS, Mouse Assay, RNA Sequencing Assay, Sequencing, Expressing, Purification

    2) Product Images from "Differential Expression of HERV-K (HML-2) Proviruses in Cells and Virions of the Teratocarcinoma Cell Line Tera-1"

    Article Title: Differential Expression of HERV-K (HML-2) Proviruses in Cells and Virions of the Teratocarcinoma Cell Line Tera-1

    Journal: Viruses

    doi: 10.3390/v7030939

    HML-2 expression in Tera-1 cells and virions. ( A , B ) RNASeq reads originating from Tera-1 cells were aligned to the hg19 build of the human genome and analyzed using the Plus stranded, Unique Only analysis, except as indicated. ( E – F ) RNASeq reads originating from Tera-1 virions were aligned to the hg19 build of the human genome and analyzed using the Unstranded, Unique Only analysis, except as indicated, due to the input library not being stranded. ( A , E ) Relative transcript expression values (FPKM) for cellular genes, total HML-2 and the most abundantly expressed or packaged HML-2 transcripts are plotted for Tera-1 cells ( A ) and Tera-1 virions ( E ). ( B , F ) Abundance of transcripts for each provirus in Tera-1 cells ( B ) and virions ( F ) is plotted according to (provirus FPKM)/(total HML-2 FPKM) × 100. Proviruses with (*) were predicted to be underrepresented by the in silico analysis, as used in Figure 1 . ( C ) Open reading frames for gag , pol and env were determined for proviruses making up 96.81% of all HML-2 reads shown in Figure 2 B. If a provirus had the potential to express open reading frame(s) (ORF(s)), the abundance of the provirus in the cell was allocated to each ORF, as this represents the maximum probability of that ORF being expressed. Splicing was not considered for this analysis. ( D ) Type 1/2 status was determined for HML-2 proviruses making up 96.81% of all HML-2 reads, listed in Figure 2 B. Unknown indicates that the entire pol - env boundary region was not present in the provirus, preventing identification of provirus type.
    Figure Legend Snippet: HML-2 expression in Tera-1 cells and virions. ( A , B ) RNASeq reads originating from Tera-1 cells were aligned to the hg19 build of the human genome and analyzed using the Plus stranded, Unique Only analysis, except as indicated. ( E – F ) RNASeq reads originating from Tera-1 virions were aligned to the hg19 build of the human genome and analyzed using the Unstranded, Unique Only analysis, except as indicated, due to the input library not being stranded. ( A , E ) Relative transcript expression values (FPKM) for cellular genes, total HML-2 and the most abundantly expressed or packaged HML-2 transcripts are plotted for Tera-1 cells ( A ) and Tera-1 virions ( E ). ( B , F ) Abundance of transcripts for each provirus in Tera-1 cells ( B ) and virions ( F ) is plotted according to (provirus FPKM)/(total HML-2 FPKM) × 100. Proviruses with (*) were predicted to be underrepresented by the in silico analysis, as used in Figure 1 . ( C ) Open reading frames for gag , pol and env were determined for proviruses making up 96.81% of all HML-2 reads shown in Figure 2 B. If a provirus had the potential to express open reading frame(s) (ORF(s)), the abundance of the provirus in the cell was allocated to each ORF, as this represents the maximum probability of that ORF being expressed. Splicing was not considered for this analysis. ( D ) Type 1/2 status was determined for HML-2 proviruses making up 96.81% of all HML-2 reads, listed in Figure 2 B. Unknown indicates that the entire pol - env boundary region was not present in the provirus, preventing identification of provirus type.

    Techniques Used: Expressing, In Silico

    HML-2 Promoter Expression in Tera-1 Cells. ( A ) Comparison of the relative transcript expression level (FPKM; black) for a provirus and its corresponding relative luciferase expression level in Tera-1 cells transfected with a vector containing a luciferase reporter gene downstream of the indicated proviral 5’ LTR. LTR activity is expressed as relative light units (RLU; gray) normalized to a control construct with a Renilla luciferase gene driven by an SV40 promoter. The relative promoter activities of the LTR Hs located 551 bp upstream from the 22q11.23 provirus, the 5’ LTR 5B of the 22q11.23 provirus and the 5’ LTR Hs of six other expressed proviruses in Tera-1 cells are shown. ( B ) Schematic of the 22q11.23 LTR Hs, showing the U3, R and U5 regions. Predicted transcriptional start sites are indicated with black arrows and nucleotide position. Colored boxes indicate previously described promoter element motifs [ 62 , 63 , 64 ]. Lines below the LTR diagram indicate the regions included in each truncated LTR construct, and numbers to the right of each line indicate the nucleotide position at which the LTR was truncated. GA, GA rich motif (nt 379–386, sequence GGGAAGGG); E, enhancer box (nt 465–476, sequence TTGCAGTTGAGA; nt 485–496, sequence AGGCATCTGTCT; nt 832–843, sequence CTCCATATGCTG); GC, GC rich motif nt 759–763, (sequence CCCCC; nt 602–606, sequence GGCGG); TATA, TATA box (nt 790–797, sequence AATAAATA); Inr, initiator element (nt 807–812, sequence CTCAGA). Cartoon is not drawn to scale. ( C ) Relative promoter expression levels of truncated 22q11.23 LTR Hs constructs in Tera-1 cells (Kruskal-Wallis, * p
    Figure Legend Snippet: HML-2 Promoter Expression in Tera-1 Cells. ( A ) Comparison of the relative transcript expression level (FPKM; black) for a provirus and its corresponding relative luciferase expression level in Tera-1 cells transfected with a vector containing a luciferase reporter gene downstream of the indicated proviral 5’ LTR. LTR activity is expressed as relative light units (RLU; gray) normalized to a control construct with a Renilla luciferase gene driven by an SV40 promoter. The relative promoter activities of the LTR Hs located 551 bp upstream from the 22q11.23 provirus, the 5’ LTR 5B of the 22q11.23 provirus and the 5’ LTR Hs of six other expressed proviruses in Tera-1 cells are shown. ( B ) Schematic of the 22q11.23 LTR Hs, showing the U3, R and U5 regions. Predicted transcriptional start sites are indicated with black arrows and nucleotide position. Colored boxes indicate previously described promoter element motifs [ 62 , 63 , 64 ]. Lines below the LTR diagram indicate the regions included in each truncated LTR construct, and numbers to the right of each line indicate the nucleotide position at which the LTR was truncated. GA, GA rich motif (nt 379–386, sequence GGGAAGGG); E, enhancer box (nt 465–476, sequence TTGCAGTTGAGA; nt 485–496, sequence AGGCATCTGTCT; nt 832–843, sequence CTCCATATGCTG); GC, GC rich motif nt 759–763, (sequence CCCCC; nt 602–606, sequence GGCGG); TATA, TATA box (nt 790–797, sequence AATAAATA); Inr, initiator element (nt 807–812, sequence CTCAGA). Cartoon is not drawn to scale. ( C ) Relative promoter expression levels of truncated 22q11.23 LTR Hs constructs in Tera-1 cells (Kruskal-Wallis, * p

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

    Transcription of HML-2 proviruses is driven by the native LTR or a nearby element. ( A ) Neighbor-joining tree of the 5’ LTR sequences of the HML-2 proviruses expressed in Tera-1 cells. The p-distance method was used to calculate distance and bootstrap values are indicated (1000 replicates). Proviruses with (*) were predicted to be underrepresented by the in silico analysis, as in Figure 1 . Solid squares (∎) indicate those proviruses (11q23.3 and 11q12.3) with minus strand transcription. Solid diamonds (♦) indicate those proviruses (4p16.3a and 22q11.23) with plus strand transcription, but which appear to originate from a neighboring transcription unit and not the corresponding 5’ LTR. ( B ) A cartoon of two proviruses located on chromosome 22 and their method of transcription. Provirus 22q11.21 (LTR Hs, FPKM = 26.11) is located 2.1 kb downstream from the expressed gene PRODH ( Pro line D e h ydrogenase (oxidase) 1, FPKM = 11.53) but in the opposite transcriptional orientation. The 5’ LTR of 22q11.21 appears to drive proviral transcription in Tera-1 cells. Provirus 22q11.23 (FPKM = 26.94) appears to be transcribed solely through the use of an LTR Hs (FPKM = 0.31) located 551 bp upstream from the provirus. This transcript coincides with an annotated lincRNA ( l arge i ntergenic n on- c oding RNA) [ 59 ]. See supplemental Figures S3 and S4 for more detail. Cartoon is not drawn to scale.
    Figure Legend Snippet: Transcription of HML-2 proviruses is driven by the native LTR or a nearby element. ( A ) Neighbor-joining tree of the 5’ LTR sequences of the HML-2 proviruses expressed in Tera-1 cells. The p-distance method was used to calculate distance and bootstrap values are indicated (1000 replicates). Proviruses with (*) were predicted to be underrepresented by the in silico analysis, as in Figure 1 . Solid squares (∎) indicate those proviruses (11q23.3 and 11q12.3) with minus strand transcription. Solid diamonds (♦) indicate those proviruses (4p16.3a and 22q11.23) with plus strand transcription, but which appear to originate from a neighboring transcription unit and not the corresponding 5’ LTR. ( B ) A cartoon of two proviruses located on chromosome 22 and their method of transcription. Provirus 22q11.21 (LTR Hs, FPKM = 26.11) is located 2.1 kb downstream from the expressed gene PRODH ( Pro line D e h ydrogenase (oxidase) 1, FPKM = 11.53) but in the opposite transcriptional orientation. The 5’ LTR of 22q11.21 appears to drive proviral transcription in Tera-1 cells. Provirus 22q11.23 (FPKM = 26.94) appears to be transcribed solely through the use of an LTR Hs (FPKM = 0.31) located 551 bp upstream from the provirus. This transcript coincides with an annotated lincRNA ( l arge i ntergenic n on- c oding RNA) [ 59 ]. See supplemental Figures S3 and S4 for more detail. Cartoon is not drawn to scale.

    Techniques Used: In Silico

    RNASeq analysis of HML-2 expression in Tera-1 cells. ( A ) RNASeq reads derived from Tera-1 cellular RNA were aligned to the hg19 build of the human genome, using either a stranded (“Plus Stranded”) or unstranded (“Unstranded”) alignment. Aligned reads were either kept in full (“Unfiltered”), or were filtered based on mapping quality scores to only retain reads that uniquely aligned to one map location (“Unique Only”). The fragments per kilobase per million mapped reads (FPKM) values representing relative expression in Tera-1 cells were determined either with a multi-read correct parameter (“Multi-read Correct”) that proportionally allocates multi-reads to mapping locations, or without this parameter. FPKM values for selected HML-2 proviruses and the cellular genes GAPDH and β-actin (ACTB) across the analyses were log-normalized and used for heatmap generation to demonstrate the effects of the different analyses on expression levels. Proviruses and gene loci are divided into four groups according to their relative values following the different analyses: stable (Group 1); decrease after Unique Only (Group 2); decrease after Plus stranded alignment (Group 3); and decrease after Unique Only and Plus stranded analysis (Group 4). Log-normalized FPKM is shown by the colors from high (red) to low (blue), as indicated in the chart to the right. The (*) symbols refer to proviruses predicted to be underrepresented by 15% or more based on an in silico simulation. ( B ) A neighbor-joining tree of the underrepresented proviruses was created using the full provirus sequence. The p-distance method was used and bootstrap values are indicated as percent of 1000 replicates. ( C ) The abundance of transcripts after the Plus stranded, Unfiltered and the Plus Stranded, Unique Only analyses are plotted against estimated times of integration to show the effect of the Unique Only analysis on recently integrated proviruses. The 0–2 mya group includes human specific integrations with high sequence similarity predicted to be underrepresented in the Unique Only RNASeq in silico simulation. The relative abundance in Tera-1 cells was calculated for each provirus based on (provirus FPKM)/(total HML-2 provirus FPKM) × 100. Elements without 5’ or 3’ LTRs were unsuitable for age estimation and are not included.
    Figure Legend Snippet: RNASeq analysis of HML-2 expression in Tera-1 cells. ( A ) RNASeq reads derived from Tera-1 cellular RNA were aligned to the hg19 build of the human genome, using either a stranded (“Plus Stranded”) or unstranded (“Unstranded”) alignment. Aligned reads were either kept in full (“Unfiltered”), or were filtered based on mapping quality scores to only retain reads that uniquely aligned to one map location (“Unique Only”). The fragments per kilobase per million mapped reads (FPKM) values representing relative expression in Tera-1 cells were determined either with a multi-read correct parameter (“Multi-read Correct”) that proportionally allocates multi-reads to mapping locations, or without this parameter. FPKM values for selected HML-2 proviruses and the cellular genes GAPDH and β-actin (ACTB) across the analyses were log-normalized and used for heatmap generation to demonstrate the effects of the different analyses on expression levels. Proviruses and gene loci are divided into four groups according to their relative values following the different analyses: stable (Group 1); decrease after Unique Only (Group 2); decrease after Plus stranded alignment (Group 3); and decrease after Unique Only and Plus stranded analysis (Group 4). Log-normalized FPKM is shown by the colors from high (red) to low (blue), as indicated in the chart to the right. The (*) symbols refer to proviruses predicted to be underrepresented by 15% or more based on an in silico simulation. ( B ) A neighbor-joining tree of the underrepresented proviruses was created using the full provirus sequence. The p-distance method was used and bootstrap values are indicated as percent of 1000 replicates. ( C ) The abundance of transcripts after the Plus stranded, Unfiltered and the Plus Stranded, Unique Only analyses are plotted against estimated times of integration to show the effect of the Unique Only analysis on recently integrated proviruses. The 0–2 mya group includes human specific integrations with high sequence similarity predicted to be underrepresented in the Unique Only RNASeq in silico simulation. The relative abundance in Tera-1 cells was calculated for each provirus based on (provirus FPKM)/(total HML-2 provirus FPKM) × 100. Elements without 5’ or 3’ LTRs were unsuitable for age estimation and are not included.

    Techniques Used: Expressing, Derivative Assay, In Silico, Sequencing

    3) Product Images from "Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding"

    Article Title: Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1977

    DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.
    Figure Legend Snippet: DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.

    Techniques Used: Infection, Clone Assay

    Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.
    Figure Legend Snippet: Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.

    Techniques Used: Plasmid Preparation, Mouse Assay, Transplantation Assay, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing

    4) Product Images from "Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine"

    Article Title: Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154949

    Fluorescence activated nuclear sorting (FANS) of three classes of visceral adipose tissue cellular nuclei immuno-stained for PPARg2. A. Histogram of PPARg2-Neg, -Low, -Med. and -High stained nuclei from sorting experiment (log scale) gated for DNA content (DAPI subset, S1B Fig ) and forward and side light scattering ( S1A Fig ). B . PPARg2 transcript levels were assayed among the four fractions of nuclei by qRT-PCR. C, D, E, F. Merged immunofluorescence microscope images of four isolated fractions of nuclei without re-staining. G. Comparison of the average nuclear area for the four fractions from one experiment (N = 100). There were statistically significant differences between all of the fractions of nuclear areas except for between PPARg2-Low and PPARg2-Neg fractions. H. PPARg2-High nuclei from image H showing the DAPI staining alone to reveal decondensed nuclei, however, some strongly stained PPARg2-High nuclei are small reflecting some heterogeneity in their morphology, as indicated by white arrows. For antibodies see S1 Table . A p value of P
    Figure Legend Snippet: Fluorescence activated nuclear sorting (FANS) of three classes of visceral adipose tissue cellular nuclei immuno-stained for PPARg2. A. Histogram of PPARg2-Neg, -Low, -Med. and -High stained nuclei from sorting experiment (log scale) gated for DNA content (DAPI subset, S1B Fig ) and forward and side light scattering ( S1A Fig ). B . PPARg2 transcript levels were assayed among the four fractions of nuclei by qRT-PCR. C, D, E, F. Merged immunofluorescence microscope images of four isolated fractions of nuclei without re-staining. G. Comparison of the average nuclear area for the four fractions from one experiment (N = 100). There were statistically significant differences between all of the fractions of nuclear areas except for between PPARg2-Low and PPARg2-Neg fractions. H. PPARg2-High nuclei from image H showing the DAPI staining alone to reveal decondensed nuclei, however, some strongly stained PPARg2-High nuclei are small reflecting some heterogeneity in their morphology, as indicated by white arrows. For antibodies see S1 Table . A p value of P

    Techniques Used: Fluorescence, Staining, Quantitative RT-PCR, Immunofluorescence, Microscopy, Isolation

    Distribution of 5hmC among adipose tissue nuclei. IFM and FNC were used to examine 5hmC levels among visceral adipose tissue nuclei. A. A field of VAT nuclei examined with various combinations of DAPI staining for DNA, and immunostaining with mouse anti-PPARg2 + goat anti-mouse Alexafluor488 and rabbit anti-5hmC + goat anti-rabbit Alexafluor633. White arrows indicate those large, decondensed nuclei that are stained strongly for both 5hmC and PPARg2. B. Flow Cytometry of VAT nuclei immunostained as in A. C. Goat anti-rabbit secondary antibody used in B shows only modest background staining of nuclei. Nuclei were gated for DAPI ( > 2C DNA content) and size and shape by light scattering as in S1 Fig . For antibodies see S1 Table .
    Figure Legend Snippet: Distribution of 5hmC among adipose tissue nuclei. IFM and FNC were used to examine 5hmC levels among visceral adipose tissue nuclei. A. A field of VAT nuclei examined with various combinations of DAPI staining for DNA, and immunostaining with mouse anti-PPARg2 + goat anti-mouse Alexafluor488 and rabbit anti-5hmC + goat anti-rabbit Alexafluor633. White arrows indicate those large, decondensed nuclei that are stained strongly for both 5hmC and PPARg2. B. Flow Cytometry of VAT nuclei immunostained as in A. C. Goat anti-rabbit secondary antibody used in B shows only modest background staining of nuclei. Nuclei were gated for DAPI ( > 2C DNA content) and size and shape by light scattering as in S1 Fig . For antibodies see S1 Table .

    Techniques Used: Staining, Immunostaining, Flow Cytometry, Cytometry

    5hmC levels vary dramatically across gene regions based on transcript levels in adipose tissue and the class of VAT nuclei. 25,321 S . scrofa genes were ranked into five quintiles (5,064 to 5,065 genes in each) based on the levels of adipose tissue transcript expression determined by RNAseq (1st quintile represents the lowest levels of RNA expression, 5th quintile the highest). A. The differences in gene region 5hmC levels for PPARg2-High, -Low, and -Neg nuclei are shown for each quintile of expressed genes. PPARg2-Neg nuclei had the lowest 5hmC levels in each quintile, while PPARg2-High nuclei had slightly higher 5hmC levels than PPARg2-Low nuclei. B. 5hmC levels are shown for all 5 quintiles for the PPARg2-High, PPARg2-Med+Low, and PPARg2-Neg nuclei. A B. 5hmC levels were lower in the gene body relative to the flanking gene regions for the two lowest quintile expression groups of transcripts, whereas for the 3 highest transcript expression groups the pattern was reversed with 5hmC levels being the highest in the gene body. Gene regions were divided into three parts: 100 kb upstream of the transcription start site (UTSS), gene body (GB, TSS to transcription stop site (TTS)), and 100 kb downstream of the TTS (DTTS).
    Figure Legend Snippet: 5hmC levels vary dramatically across gene regions based on transcript levels in adipose tissue and the class of VAT nuclei. 25,321 S . scrofa genes were ranked into five quintiles (5,064 to 5,065 genes in each) based on the levels of adipose tissue transcript expression determined by RNAseq (1st quintile represents the lowest levels of RNA expression, 5th quintile the highest). A. The differences in gene region 5hmC levels for PPARg2-High, -Low, and -Neg nuclei are shown for each quintile of expressed genes. PPARg2-Neg nuclei had the lowest 5hmC levels in each quintile, while PPARg2-High nuclei had slightly higher 5hmC levels than PPARg2-Low nuclei. B. 5hmC levels are shown for all 5 quintiles for the PPARg2-High, PPARg2-Med+Low, and PPARg2-Neg nuclei. A B. 5hmC levels were lower in the gene body relative to the flanking gene regions for the two lowest quintile expression groups of transcripts, whereas for the 3 highest transcript expression groups the pattern was reversed with 5hmC levels being the highest in the gene body. Gene regions were divided into three parts: 100 kb upstream of the transcription start site (UTSS), gene body (GB, TSS to transcription stop site (TTS)), and 100 kb downstream of the TTS (DTTS).

    Techniques Used: Expressing, RNA Expression

    5) Product Images from "Faster embryonic segmentation through elevated Delta-Notch signalling"

    Article Title: Faster embryonic segmentation through elevated Delta-Notch signalling

    Journal: Nature Communications

    doi: 10.1038/ncomms11861

    A range of transgenic lines with elevated spatiotemporally accurate DeltaD expression. ( a – f ) deltaD-venus recapitulates WT deltaD expression pattern. Whole-mount ISH, 9 somite-stage embryos lateral view with either deltaD or egfp anti-sense riboprobe in deltaD mutant after eight ( aei ) +/− ( a ), WT AB ( b ) or deltaD-venus transgenic lines ( d – f ). ( c ) Schematic representation of deltaD-venus construct for generating transgenic lines. See Supplementary Fig. 1 . ( a ) Scale bar, 300 μm. ( f – h ) deltaD transgenic copy numbers determined by quantitative real-time PCR from genomic DNA. Copy number is insensitive to aei point mutation allele ( g ). Transgenic copies ( h , i ) are total deltaD copies minus 2 endogenous copies. Empty bars, heterozygous genotypes; black bars, homozygous genotypes. Data pooled from ⩾3 independent experiments, mean±s.d. ( j ) DeltaD protein expression visualized and quantitated by immunostaining. (ja–jc) PSM of flat-mounted 9 somite-stage embryos, circle shows region used for intensity measurement. Scale bar, 50 μm. ( k ) DeltaD protein expression levels versus deltaD gene copy number. Expression level in aei −/− was defined as 0. Grey dashed line, linear fit as the formula and R 2 value at upper left corner. Inset shows magnification of data points at origin. Mean±s.d. ( n ⩾5).
    Figure Legend Snippet: A range of transgenic lines with elevated spatiotemporally accurate DeltaD expression. ( a – f ) deltaD-venus recapitulates WT deltaD expression pattern. Whole-mount ISH, 9 somite-stage embryos lateral view with either deltaD or egfp anti-sense riboprobe in deltaD mutant after eight ( aei ) +/− ( a ), WT AB ( b ) or deltaD-venus transgenic lines ( d – f ). ( c ) Schematic representation of deltaD-venus construct for generating transgenic lines. See Supplementary Fig. 1 . ( a ) Scale bar, 300 μm. ( f – h ) deltaD transgenic copy numbers determined by quantitative real-time PCR from genomic DNA. Copy number is insensitive to aei point mutation allele ( g ). Transgenic copies ( h , i ) are total deltaD copies minus 2 endogenous copies. Empty bars, heterozygous genotypes; black bars, homozygous genotypes. Data pooled from ⩾3 independent experiments, mean±s.d. ( j ) DeltaD protein expression visualized and quantitated by immunostaining. (ja–jc) PSM of flat-mounted 9 somite-stage embryos, circle shows region used for intensity measurement. Scale bar, 50 μm. ( k ) DeltaD protein expression levels versus deltaD gene copy number. Expression level in aei −/− was defined as 0. Grey dashed line, linear fit as the formula and R 2 value at upper left corner. Inset shows magnification of data points at origin. Mean±s.d. ( n ⩾5).

    Techniques Used: Transgenic Assay, Expressing, In Situ Hybridization, Mutagenesis, Construct, Real-time Polymerase Chain Reaction, Immunostaining

    6) Product Images from "c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart"

    Article Title: c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart

    Journal: Nature

    doi: 10.1038/nature13309

    Analysis of c-kit lineage labeling in the heart at P0 (birth) a, Diagram of the timing whereby newborn Kit +/Cre × R-GFP mice were analyzed for all subsequent experiments in this figure. b, Histological sections for eGFP fluorescence (green) from the ileum and lung at P0 showing the characteristic c-kit labeling pattern as observed at other time points or in other studies when antibodies were employed. Blue shows nuclei c, Histological section for eGFP fluorescence (green) from the heart at P0. Blue shows nuclei and magnification was 40X. d, Immunohistochemical tissue section from the P0 heart of Kit +/Cre × R-GFP mice stained with sarcomeric α-actin (red) to show all underlying cardiomyocytes (right panel) or with eGFP expression in green (left panel) as being c-kit derived. The green cells noted by the arrows are non-myocytes that do not express sarcomeric α-actin. e, eGFP expression alone (left) or eGFP with co-staining for cardiomyocytes in red (sarcomeric α-actin) from heart sections at P0 of Kit +/Cre × R-GFP mice. Blue staining depicts nuclei. The cardiomyocyte that is shown has clear striations in the eGFP staining pattern, while the 2 non-myocytes do not show striated eGFP and also lack sarcomeric α-actin staining. f, eGFP expression alone in green (left) with nuclei in blue or eGFP with sarcomeric α-actin co-staining (red) from heart sections at P0 of Kit +/Cre × R-GFP mice. All eGFP + cells shown lack striations and are non-myocytes although the 2 cells in the center sit directly on top of cardiomyocytes and could be easily mis-interpreted. Great care is needed in scoring myocytes in the P0 heart because they are small and often the same size as eGFP + non-myocytes. g, eGFP expression (green) with nuclei in blue and cardiomyocytes identified in red with sarcomeric α-actin antibody from heart histological sections at P0 of Kit +/Cre × R-GFP mice. Here the data show c-kit lineage derived cardiomyocytes that appear in a loose cluster (arrows), presumably from a clonal expansion event earlier in development.
    Figure Legend Snippet: Analysis of c-kit lineage labeling in the heart at P0 (birth) a, Diagram of the timing whereby newborn Kit +/Cre × R-GFP mice were analyzed for all subsequent experiments in this figure. b, Histological sections for eGFP fluorescence (green) from the ileum and lung at P0 showing the characteristic c-kit labeling pattern as observed at other time points or in other studies when antibodies were employed. Blue shows nuclei c, Histological section for eGFP fluorescence (green) from the heart at P0. Blue shows nuclei and magnification was 40X. d, Immunohistochemical tissue section from the P0 heart of Kit +/Cre × R-GFP mice stained with sarcomeric α-actin (red) to show all underlying cardiomyocytes (right panel) or with eGFP expression in green (left panel) as being c-kit derived. The green cells noted by the arrows are non-myocytes that do not express sarcomeric α-actin. e, eGFP expression alone (left) or eGFP with co-staining for cardiomyocytes in red (sarcomeric α-actin) from heart sections at P0 of Kit +/Cre × R-GFP mice. Blue staining depicts nuclei. The cardiomyocyte that is shown has clear striations in the eGFP staining pattern, while the 2 non-myocytes do not show striated eGFP and also lack sarcomeric α-actin staining. f, eGFP expression alone in green (left) with nuclei in blue or eGFP with sarcomeric α-actin co-staining (red) from heart sections at P0 of Kit +/Cre × R-GFP mice. All eGFP + cells shown lack striations and are non-myocytes although the 2 cells in the center sit directly on top of cardiomyocytes and could be easily mis-interpreted. Great care is needed in scoring myocytes in the P0 heart because they are small and often the same size as eGFP + non-myocytes. g, eGFP expression (green) with nuclei in blue and cardiomyocytes identified in red with sarcomeric α-actin antibody from heart histological sections at P0 of Kit +/Cre × R-GFP mice. Here the data show c-kit lineage derived cardiomyocytes that appear in a loose cluster (arrows), presumably from a clonal expansion event earlier in development.

    Techniques Used: Labeling, Mouse Assay, Fluorescence, Immunohistochemistry, Staining, Expressing, Derivative Assay

    Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formation a, Genetic cross between Kit +/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g ). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit +/MCM × R-GFP mice without ( c ) or with tamoxifen ( d ). e, FACS quantification of eGFP + cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit +/MCM × R-GFP). *p
    Figure Legend Snippet: Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formation a, Genetic cross between Kit +/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g ). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit +/MCM × R-GFP mice without ( c ) or with tamoxifen ( d ). e, FACS quantification of eGFP + cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit +/MCM × R-GFP). *p

    Techniques Used: Expressing, Mouse Assay, FACS

    Identification of non-myocytes from the hearts of Kit +/Cre × R-GFP mice Kit +/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit -Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP + and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit +/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP + (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP + non-myocytes, which in panel c , were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.
    Figure Legend Snippet: Identification of non-myocytes from the hearts of Kit +/Cre × R-GFP mice Kit +/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit -Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP + and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit +/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP + (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP + non-myocytes, which in panel c , were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.

    Techniques Used: Mouse Assay, Labeling, Immunohistochemistry, Expressing, Staining, Marker, Isolation, Immunocytochemistry

    Verifying the extent of eGFP + cardiomyocytes by an independent laboratory from blinded histological heart samples Unprocessed cryosections and paraffin sections from the hearts of Kit +/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP + myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP + cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP + cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP + expressing cardiomyocyte and the arrowheads show eGFP + non-myocytes.
    Figure Legend Snippet: Verifying the extent of eGFP + cardiomyocytes by an independent laboratory from blinded histological heart samples Unprocessed cryosections and paraffin sections from the hearts of Kit +/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP + myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP + cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP + cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP + expressing cardiomyocyte and the arrowheads show eGFP + non-myocytes.

    Techniques Used: Mouse Assay, Negative Control, Staining, Fluorescence, Labeling, Expressing

    Assessing cardiomyocyte differentiation markers from total non-myocytes in the heart Adult cardiac interstitial cells isolated from a Kit +/Cre × R-GFP mouse were treated with dexamethasone for 1 week. Cells were then fixed and subjected to immunocytochemistry for the indicated antibodies. c-kit lineage derived cells were green (eGFP + ) and showed fluorescence in the cytosol and nucleus. The data show eGFP cells that express markers of differentiated cardiomyocytes such as α-actinin, troponin T, and the transcription factor GATA4 (all in red) but not the fibroblast marker vimentin (white), nuclei were stained blue (right panels). These results indicate that eGFP + Kit -Cre expressing cells can generate pre-differentiated cardiomyocytes as well as non-eGFP interstitial cells; hence the cells identified by the Kit -Cre (knock-in) reporter strategy are representative of how endogenous c-kit + expressing cells truly function.
    Figure Legend Snippet: Assessing cardiomyocyte differentiation markers from total non-myocytes in the heart Adult cardiac interstitial cells isolated from a Kit +/Cre × R-GFP mouse were treated with dexamethasone for 1 week. Cells were then fixed and subjected to immunocytochemistry for the indicated antibodies. c-kit lineage derived cells were green (eGFP + ) and showed fluorescence in the cytosol and nucleus. The data show eGFP cells that express markers of differentiated cardiomyocytes such as α-actinin, troponin T, and the transcription factor GATA4 (all in red) but not the fibroblast marker vimentin (white), nuclei were stained blue (right panels). These results indicate that eGFP + Kit -Cre expressing cells can generate pre-differentiated cardiomyocytes as well as non-eGFP interstitial cells; hence the cells identified by the Kit -Cre (knock-in) reporter strategy are representative of how endogenous c-kit + expressing cells truly function.

    Techniques Used: Isolation, Immunocytochemistry, Derivative Assay, Fluorescence, Marker, Staining, Expressing, Knock-In

    Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit +/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit +MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across > 50 histological sections of all eGFP + expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P
    Figure Legend Snippet: Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit +/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit +MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across > 50 histological sections of all eGFP + expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P

    Techniques Used: Mouse Assay, Quantitation Assay, Expressing

    Analysis of eGFP + myocytes in the hearts of Kit +/MCM × R-GFP mice after isoproterenol infusion-induced injury a, Schematic diagram showing tamoxifen treatment of Kit +/MCM × R-GFP mice between 7 and 14 weeks of age with isoproterenol (ISO) infusion occurring between weeks 10–14. b, c, Quantitation and imaging of disassociated cardiomyocytes (separate images shown at 2 different magnifications) from the hearts of ISO injured Kit +/MCM × R-GFP mice, which showed rare but definitive cardiomyocyte labeling. *P
    Figure Legend Snippet: Analysis of eGFP + myocytes in the hearts of Kit +/MCM × R-GFP mice after isoproterenol infusion-induced injury a, Schematic diagram showing tamoxifen treatment of Kit +/MCM × R-GFP mice between 7 and 14 weeks of age with isoproterenol (ISO) infusion occurring between weeks 10–14. b, c, Quantitation and imaging of disassociated cardiomyocytes (separate images shown at 2 different magnifications) from the hearts of ISO injured Kit +/MCM × R-GFP mice, which showed rare but definitive cardiomyocyte labeling. *P

    Techniques Used: Mouse Assay, Quantitation Assay, Imaging, Labeling

    Quantitation of Cre activity and DNA recombination in the hearts of Kit +/MCM × R-GFP mice a, Time line for tamoxifen administration in Kit +/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit +/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n =3 Kit +/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit +/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit +/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP + cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.
    Figure Legend Snippet: Quantitation of Cre activity and DNA recombination in the hearts of Kit +/MCM × R-GFP mice a, Time line for tamoxifen administration in Kit +/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit +/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n =3 Kit +/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit +/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit +/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP + cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.

    Techniques Used: Quantitation Assay, Activity Assay, Mouse Assay, Polymerase Chain Reaction, Generated, Purification, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    7) Product Images from "Characterization and metabolic synthetic lethal testing in a new model of SDH-loss familial pheochromocytoma and paraganglioma"

    Article Title: Characterization and metabolic synthetic lethal testing in a new model of SDH-loss familial pheochromocytoma and paraganglioma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23639

    Analysis of genome-wide methylation patterns in SDHC-loss iMEFs ( A ) Volcano plot showing CpG site mean methylation difference versus –log( p -value) from RnBeads differential methylation analysis. Comparison was generated between control time series specimens and experimental specimens, excluding day 0. Blue dots correspond to the top 0.1 quantile of the dataset, as quantified by methylation combined rank. ( B ) Unsupervised hierarchical clustering of samples based upon CpG site DNA methylation patterns. The data used for clustering includes the top 0.1 quantile of CpG sites, ordered by methylation combined rank. ( C ) Correlation heat maps showing the emergence of CpG site DNA methylation difference between experimental and control iMEF lines following induction of SDHC loss with doxycycline. Only differences emerging after day 0 are shown. Colors correspond to data point density (red: high; green: intermediate; blue: low). ( D ) Time course analysis of CpG site methylation change, separated according to day 0 methylation status. Left histograms illustrate change in DNA methylation for subset of CpG sites with little initial methylation (day 0 beta value
    Figure Legend Snippet: Analysis of genome-wide methylation patterns in SDHC-loss iMEFs ( A ) Volcano plot showing CpG site mean methylation difference versus –log( p -value) from RnBeads differential methylation analysis. Comparison was generated between control time series specimens and experimental specimens, excluding day 0. Blue dots correspond to the top 0.1 quantile of the dataset, as quantified by methylation combined rank. ( B ) Unsupervised hierarchical clustering of samples based upon CpG site DNA methylation patterns. The data used for clustering includes the top 0.1 quantile of CpG sites, ordered by methylation combined rank. ( C ) Correlation heat maps showing the emergence of CpG site DNA methylation difference between experimental and control iMEF lines following induction of SDHC loss with doxycycline. Only differences emerging after day 0 are shown. Colors correspond to data point density (red: high; green: intermediate; blue: low). ( D ) Time course analysis of CpG site methylation change, separated according to day 0 methylation status. Left histograms illustrate change in DNA methylation for subset of CpG sites with little initial methylation (day 0 beta value

    Techniques Used: Genome Wide, Methylation, Generated, DNA Methylation Assay

    Genetic and phenotypic characterization of SDHC-loss iMEFs ( A ) PCR analysis of Sdhc gene rearrangement using primers flanking [floxed] exon 4, resulting in production of a shortened PCR product upon Cre-mediated gene rearrangement. ( B ) Western blot analysis of SDHC and SDHB protein loss following Sdhc gene rearrangement. ( C ) Western blot quantitation. Colors indicate respective iMEF line (red, experimental; black, control). Symbols correspond to quantified protein (circles: SDHC; triangles: SDHB). Welch two-sample t -test of SDHC protein amount at day 12 quantified for experimental and control lines yields p -value of 0.004 ( N = 6 experimental replicates). Similar statistical analysis of SDHB protein amount yields p -value of 3E-5. ( D ) Exponential decay models of Sdhc gene rearrangement and protein loss. DNA rearrangement and SDHC protein half-lives are 1.76 and 2.17 d, respectively. Midpoints for DNA rearrangement and SDHC protein loss curves occur at 1.8 and 3.6 d, respectively. ( E ) Measured intracellular succinate abundance. Values are normalized to total protein. ( F ) Quantitation of cell population doubling time. Welch two-sample t -test of doubling time difference between experimental and control lines at day 22 yields a p -value of 0.004.
    Figure Legend Snippet: Genetic and phenotypic characterization of SDHC-loss iMEFs ( A ) PCR analysis of Sdhc gene rearrangement using primers flanking [floxed] exon 4, resulting in production of a shortened PCR product upon Cre-mediated gene rearrangement. ( B ) Western blot analysis of SDHC and SDHB protein loss following Sdhc gene rearrangement. ( C ) Western blot quantitation. Colors indicate respective iMEF line (red, experimental; black, control). Symbols correspond to quantified protein (circles: SDHC; triangles: SDHB). Welch two-sample t -test of SDHC protein amount at day 12 quantified for experimental and control lines yields p -value of 0.004 ( N = 6 experimental replicates). Similar statistical analysis of SDHB protein amount yields p -value of 3E-5. ( D ) Exponential decay models of Sdhc gene rearrangement and protein loss. DNA rearrangement and SDHC protein half-lives are 1.76 and 2.17 d, respectively. Midpoints for DNA rearrangement and SDHC protein loss curves occur at 1.8 and 3.6 d, respectively. ( E ) Measured intracellular succinate abundance. Values are normalized to total protein. ( F ) Quantitation of cell population doubling time. Welch two-sample t -test of doubling time difference between experimental and control lines at day 22 yields a p -value of 0.004.

    Techniques Used: Polymerase Chain Reaction, Western Blot, Quantitation Assay

    8) Product Images from "Communication between viruses guides lysis-lysogeny decisions"

    Article Title: Communication between viruses guides lysis-lysogeny decisions

    Journal: Nature

    doi: 10.1038/nature21049

    DNA binding and transcription regulation in the arbitrium system. (A) ChIP-seq of His-tagged AimR 15 minutes post-infection with or without 1 µM of SAIRGA peptide. Shown is the ratio, along the phage genome, between sequenced pulled-down DNA during infection without the peptide and DNA pulled-down when the peptide was present in the medium. (B) Same as panel A, shown is a zoomed-in region in the phage genome. (C) Gel-filtration results of purified AimR with or without the presence of either SAIRGA or GMPRGA peptide. Inset presents a calibration curve for the gel filtration using proteins of known sizes. (D) Expression of the AimX gene during infection. Data presented for individual biological replicates (E-G) RNA-seq coverage of the arbitrium locus at 5 minutes (E), 10 minutes (F) and 20 minutes (G) post infection. (H) Growth curves of WT and dCas9-silenced bacterial strains during phi3T-infection. Strains were infected at t=0 at MOI=0.1. Shown is average of 3 biological replicates, each with 3 technical replicates; error bars represent SE.
    Figure Legend Snippet: DNA binding and transcription regulation in the arbitrium system. (A) ChIP-seq of His-tagged AimR 15 minutes post-infection with or without 1 µM of SAIRGA peptide. Shown is the ratio, along the phage genome, between sequenced pulled-down DNA during infection without the peptide and DNA pulled-down when the peptide was present in the medium. (B) Same as panel A, shown is a zoomed-in region in the phage genome. (C) Gel-filtration results of purified AimR with or without the presence of either SAIRGA or GMPRGA peptide. Inset presents a calibration curve for the gel filtration using proteins of known sizes. (D) Expression of the AimX gene during infection. Data presented for individual biological replicates (E-G) RNA-seq coverage of the arbitrium locus at 5 minutes (E), 10 minutes (F) and 20 minutes (G) post infection. (H) Growth curves of WT and dCas9-silenced bacterial strains during phi3T-infection. Strains were infected at t=0 at MOI=0.1. Shown is average of 3 biological replicates, each with 3 technical replicates; error bars represent SE.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Infection, Filtration, Purification, Expressing, RNA Sequencing Assay

    Effect of conditioned media on the infection dynamics of phage phi3T. (A) Preparation protocol of control and conditioned media. (B) Growth curves of B. subtilis 168 infected by phi3T at MOI=0.1, in control and conditioned media. (C) Growth curves of B. subtilis strain 3610 (WT) and its derivative DS4979 ( oppD ::kan) infected by phi3T at MOI=0.1. For panels B-C, data represents average of 3 biological replicates, each with 3 technical replicates; error bars represent SE. (D) Semi-quantitative PCR assay for phage lysogeny during an infection time course of B. subtilis 168 with phi3T. “No DNA”, control without DNA; “WT”, DNA from uninfected culture; “Lysogen”, genomic DNA of a phi3T lysogen .
    Figure Legend Snippet: Effect of conditioned media on the infection dynamics of phage phi3T. (A) Preparation protocol of control and conditioned media. (B) Growth curves of B. subtilis 168 infected by phi3T at MOI=0.1, in control and conditioned media. (C) Growth curves of B. subtilis strain 3610 (WT) and its derivative DS4979 ( oppD ::kan) infected by phi3T at MOI=0.1. For panels B-C, data represents average of 3 biological replicates, each with 3 technical replicates; error bars represent SE. (D) Semi-quantitative PCR assay for phage lysogeny during an infection time course of B. subtilis 168 with phi3T. “No DNA”, control without DNA; “WT”, DNA from uninfected culture; “Lysogen”, genomic DNA of a phi3T lysogen .

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    9) Product Images from "Nucleoporin 107, 62 and 153 mediate Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells"

    Article Title: Nucleoporin 107, 62 and 153 mediate Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05208-2

    Nucleoporin depletion disrupted Kcnq1ot1 ncRNA expression. a Real-time Kcnq1ot 1 ncRNA expression levels normalized to Gapdh ( n = 3 biological samples with four technical replicates per sample). b Allelic Kcnq1ot1 ncRNA expression in control and Nup -depleted XEN cells ( n = 3 biological samples; n = 4 technical replicates per sample). c Absolute allelic Kcnq1ot 1 transcript abundance determined by droplet digital PCR in control and Nup -depleted XEN cells, as measured by RNA copies µg −1 ( n = 3 biological samples). Center lines, medians; box limits, 25th and 75th percentiles as determined by R software; whiskers, 1.5 times the interquartile range from 25th and 75th percentiles; B6/maternal, red; CAST/paternal, blue; error bars, s.e.m.; *, significance p
    Figure Legend Snippet: Nucleoporin depletion disrupted Kcnq1ot1 ncRNA expression. a Real-time Kcnq1ot 1 ncRNA expression levels normalized to Gapdh ( n = 3 biological samples with four technical replicates per sample). b Allelic Kcnq1ot1 ncRNA expression in control and Nup -depleted XEN cells ( n = 3 biological samples; n = 4 technical replicates per sample). c Absolute allelic Kcnq1ot 1 transcript abundance determined by droplet digital PCR in control and Nup -depleted XEN cells, as measured by RNA copies µg −1 ( n = 3 biological samples). Center lines, medians; box limits, 25th and 75th percentiles as determined by R software; whiskers, 1.5 times the interquartile range from 25th and 75th percentiles; B6/maternal, red; CAST/paternal, blue; error bars, s.e.m.; *, significance p

    Techniques Used: Expressing, Digital PCR, Software

    Nucleoporin depletion disrupted Kcnq1ot1 ncRNA volume and Kcnq1ot1 domain positioning at the nuclear periphery. a Representative confocal nuclear images displaying Kcnq1ot1 DNA (magenta) Kcnq1ot1 ncRNA (cyan) and DAPI staining (blue) for G1-synchronized control and Nup -depleted XEN cells ( n = 4; cell number = 109–123); upper panel, DNA FISH; middle panel, RNA FISH; lower panel, merge; M maternal domain, P paternal domain; white dashed line denotes nuclear rim. In these images, red and green fluorescence was converted to magenta and cyan. b , c Percent of cells with paternal or maternal Kcnq1ot1 ncRNA signals. d Percent of cells with Kcnq1ot1 ncRNA signal volume; low, 0–0.7 μm 3 ; medium, 0.7–1.4 μm 3 ; high, 1.4–2.1 μm 3 ; very high, > 2.1 μm 3 . e Distance of the paternal and maternal Kcnq1ot1 domain from the nuclear membrane in control and Nup -depleted XEN cells. The maternal Kcnq1ot1 domain was randomly positioned within the nucleus (expected nuclear periphery (NP) 15%; subnuclear periphery (SP) 30%; nuclear interior (NI) 60%), except for Nup153 -depleted cells with a Kcnq1ot1 ncRNA signal (si153 M+). For these analyses, cells with no RNA but detectable DNA FISH signals were included, while those lacking DNA signals were excluded. NP, 0–0.5 μm; SP, 0.5–1.5 μm; NI, 1.5–4 μm; error bars, s.e.m.; *, significance p
    Figure Legend Snippet: Nucleoporin depletion disrupted Kcnq1ot1 ncRNA volume and Kcnq1ot1 domain positioning at the nuclear periphery. a Representative confocal nuclear images displaying Kcnq1ot1 DNA (magenta) Kcnq1ot1 ncRNA (cyan) and DAPI staining (blue) for G1-synchronized control and Nup -depleted XEN cells ( n = 4; cell number = 109–123); upper panel, DNA FISH; middle panel, RNA FISH; lower panel, merge; M maternal domain, P paternal domain; white dashed line denotes nuclear rim. In these images, red and green fluorescence was converted to magenta and cyan. b , c Percent of cells with paternal or maternal Kcnq1ot1 ncRNA signals. d Percent of cells with Kcnq1ot1 ncRNA signal volume; low, 0–0.7 μm 3 ; medium, 0.7–1.4 μm 3 ; high, 1.4–2.1 μm 3 ; very high, > 2.1 μm 3 . e Distance of the paternal and maternal Kcnq1ot1 domain from the nuclear membrane in control and Nup -depleted XEN cells. The maternal Kcnq1ot1 domain was randomly positioned within the nucleus (expected nuclear periphery (NP) 15%; subnuclear periphery (SP) 30%; nuclear interior (NI) 60%), except for Nup153 -depleted cells with a Kcnq1ot1 ncRNA signal (si153 M+). For these analyses, cells with no RNA but detectable DNA FISH signals were included, while those lacking DNA signals were excluded. NP, 0–0.5 μm; SP, 0.5–1.5 μm; NI, 1.5–4 μm; error bars, s.e.m.; *, significance p

    Techniques Used: Staining, Fluorescence In Situ Hybridization, Fluorescence

    Nup107, Nup62 , and Nup153 depletion reactivate a subset of paternal alleles at the Kcnq1ot1 domain. Absolute allelic transcript abundance of imprinted genes determined by droplet digital PCR in control and Nup -depleted XEN cells, as a measure of RNA copies µg −1 ( n = 3 biological samples). Center lines, medians; box limits, 25th and 75th percentiles as determined by R software; whiskers, 1.5 times the interquartile range from 25th and 75th percentiles; Mat, maternal; Pat, paternal; Pat R, reactivated paternally silent allele; *, significance p
    Figure Legend Snippet: Nup107, Nup62 , and Nup153 depletion reactivate a subset of paternal alleles at the Kcnq1ot1 domain. Absolute allelic transcript abundance of imprinted genes determined by droplet digital PCR in control and Nup -depleted XEN cells, as a measure of RNA copies µg −1 ( n = 3 biological samples). Center lines, medians; box limits, 25th and 75th percentiles as determined by R software; whiskers, 1.5 times the interquartile range from 25th and 75th percentiles; Mat, maternal; Pat, paternal; Pat R, reactivated paternally silent allele; *, significance p

    Techniques Used: Digital PCR, Software

    10) Product Images from "Testosterone is an endogenous regulator of BAFF and splenic B cell number"

    Article Title: Testosterone is an endogenous regulator of BAFF and splenic B cell number

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04408-0

    Testosterone regulates splenic B cell number. a Representative plots of CD19 + CD93 + transitional B cells (tB) and CD19 + CD93 – mature B cells (matB) in the spleen from male control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) mice. b Total CD19 + B cells in the spleen in control ( n = 10) and G-ARKO ( n = 9) mice. c Total CD19 + B cells in the spleen in castrated (ORX) control and G-ARKO male mice treated with placebo (P) or testosterone (T) (25 μg/day) for 4 weeks (Control ORX P, n = 6; Control ORX T, n = 7; G-ARKO ORX P, n = 5; G-ARKO ORX T, n = 6; P -value from Kruskal–Wallis test followed by Mann–Whitney test). d % B1a (CD19 + IgM + CD43 + CD5 + ) and B1b (CD19 + IgM + CD43 + CD5 – ) cells in peritoneal fluid from control and G-ARKO male mice, n = 9/group. e–j CD19 + B cells were divided into subpopulations; graphs show transitional T1 (CD19 + CD93 + IgM + CD23 – ) B cells ( e ), transitional T2 (CD19 + CD93 + IgM hi CD23 + ) B cells ( f ), transitional T3 (CD19 + CD93 + IgM lo CD23 + ) B cells ( g ), follicular (FO) (CD19 + CD93 + CD21 int CD23 + ) B cells ( h ), marginal zone (MZ) (CD19 + CD93 + CD21 hi CD23 – ) B cells ( i ), and B1 (CD19 + CD43 + ) B cells ( j ) in the spleen in control ( n = 10) and G-ARKO ( n = 9) mice. k Sections of spleens from control and G-ARKO male mice. green, B cells (B220); red, T cells (TCRβ); and blue, metallophilic macrophages (MOMA/CD169). Scale bar = 500 μm. l–n Number of follicles per section ( l ) and mean B cell area ( m ) and mean PALS area ( n ) per follicle in spleen sections from control and G-ARKO mice, n = 4/group. o Total IgG levels in serum from control ( n = 22) and G-ARKO ( n = 19) mice. p IgG autoantibodies against DNA in control ( n = 23) and G-ARKO ( n = 18) mice. All bars indicate means; circles represent individual mice. * P
    Figure Legend Snippet: Testosterone regulates splenic B cell number. a Representative plots of CD19 + CD93 + transitional B cells (tB) and CD19 + CD93 – mature B cells (matB) in the spleen from male control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) mice. b Total CD19 + B cells in the spleen in control ( n = 10) and G-ARKO ( n = 9) mice. c Total CD19 + B cells in the spleen in castrated (ORX) control and G-ARKO male mice treated with placebo (P) or testosterone (T) (25 μg/day) for 4 weeks (Control ORX P, n = 6; Control ORX T, n = 7; G-ARKO ORX P, n = 5; G-ARKO ORX T, n = 6; P -value from Kruskal–Wallis test followed by Mann–Whitney test). d % B1a (CD19 + IgM + CD43 + CD5 + ) and B1b (CD19 + IgM + CD43 + CD5 – ) cells in peritoneal fluid from control and G-ARKO male mice, n = 9/group. e–j CD19 + B cells were divided into subpopulations; graphs show transitional T1 (CD19 + CD93 + IgM + CD23 – ) B cells ( e ), transitional T2 (CD19 + CD93 + IgM hi CD23 + ) B cells ( f ), transitional T3 (CD19 + CD93 + IgM lo CD23 + ) B cells ( g ), follicular (FO) (CD19 + CD93 + CD21 int CD23 + ) B cells ( h ), marginal zone (MZ) (CD19 + CD93 + CD21 hi CD23 – ) B cells ( i ), and B1 (CD19 + CD43 + ) B cells ( j ) in the spleen in control ( n = 10) and G-ARKO ( n = 9) mice. k Sections of spleens from control and G-ARKO male mice. green, B cells (B220); red, T cells (TCRβ); and blue, metallophilic macrophages (MOMA/CD169). Scale bar = 500 μm. l–n Number of follicles per section ( l ) and mean B cell area ( m ) and mean PALS area ( n ) per follicle in spleen sections from control and G-ARKO mice, n = 4/group. o Total IgG levels in serum from control ( n = 22) and G-ARKO ( n = 19) mice. p IgG autoantibodies against DNA in control ( n = 23) and G-ARKO ( n = 18) mice. All bars indicate means; circles represent individual mice. * P

    Techniques Used: Knock-Out, Mouse Assay, MANN-WHITNEY

    11) Product Images from "TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells"

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-017-0002-y

    TKO hESCs show hypermethylation at the PAX6 P0 bivalent promoter a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using MassArray and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P
    Figure Legend Snippet: TKO hESCs show hypermethylation at the PAX6 P0 bivalent promoter a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using MassArray and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P

    Techniques Used: Binding Assay

    12) Product Images from "N6-methyladenine DNA Modification in Glioblastoma"

    Article Title: N6-methyladenine DNA Modification in Glioblastoma

    Journal: Cell

    doi: 10.1016/j.cell.2018.10.006

    Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P
    Figure Legend Snippet: Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P

    Techniques Used: Modification, Dot Blot, Derivative Assay, Mass Spectrometry

    ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P
    Figure Legend Snippet: ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P

    Techniques Used: In Vitro, Recombinant, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Standard Deviation

    13) Product Images from "Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT"

    Article Title: Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.9b00444

    Dose-dependent responses of MEFs to MNU treatment with and without the MGMT inhibitor, AA-CW236. Panel A: effect of MNU and MNU plus AA-CW236 on MEF survival. Panel B: 6-TG r mutation frequency of MEFs with and without AA-CW236 using the gpt locus as the target for mutagenicity. Panel C: levels of m6G in the DNA of gpt delta MEFs treated with MNU, with or without AA-CW236 treatment to inactivate the MGMT protein. Error bars indicate mean ± SD; there were 4–6 replicates for each dose. If an asterisk is present over a point, that point is statistically different from the point with which it is vertically aligned (P
    Figure Legend Snippet: Dose-dependent responses of MEFs to MNU treatment with and without the MGMT inhibitor, AA-CW236. Panel A: effect of MNU and MNU plus AA-CW236 on MEF survival. Panel B: 6-TG r mutation frequency of MEFs with and without AA-CW236 using the gpt locus as the target for mutagenicity. Panel C: levels of m6G in the DNA of gpt delta MEFs treated with MNU, with or without AA-CW236 treatment to inactivate the MGMT protein. Error bars indicate mean ± SD; there were 4–6 replicates for each dose. If an asterisk is present over a point, that point is statistically different from the point with which it is vertically aligned (P

    Techniques Used: Mutagenesis

    Experimental system and workflow. Embryos from the gpt delta C57BL/6J mouse were converted into a MEF cell line by lentivirus-mediated transformation. MEFs were treated with MNU alone, or with MNU plus AA-CW236, an inhibitor of the MGMT repair protein. Cellular toxicity and mutagenicity were determined. Analytical measurement of m6G, a mutagenic DNA adduct of MNU, was performed at a dose of MNU that showed a strong mutagenic response (500 μM). Mutation distributions were determined in all possible 3-base contexts. The mutational patterns generated were then compared by cosine similarity with mutational signatures derived from sequencing of human tumors.
    Figure Legend Snippet: Experimental system and workflow. Embryos from the gpt delta C57BL/6J mouse were converted into a MEF cell line by lentivirus-mediated transformation. MEFs were treated with MNU alone, or with MNU plus AA-CW236, an inhibitor of the MGMT repair protein. Cellular toxicity and mutagenicity were determined. Analytical measurement of m6G, a mutagenic DNA adduct of MNU, was performed at a dose of MNU that showed a strong mutagenic response (500 μM). Mutation distributions were determined in all possible 3-base contexts. The mutational patterns generated were then compared by cosine similarity with mutational signatures derived from sequencing of human tumors.

    Techniques Used: Transformation Assay, Mutagenesis, Generated, Derivative Assay, Sequencing

    14) Product Images from "Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT"

    Article Title: Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.9b00444

    Dose-dependent responses of MEFs to MNU treatment with and without the MGMT inhibitor, AA-CW236. Panel A: effect of MNU and MNU plus AA-CW236 on MEF survival. Panel B: 6-TG r mutation frequency of MEFs with and without AA-CW236 using the gpt locus as the target for mutagenicity. Panel C: levels of m6G in the DNA of gpt delta MEFs treated with MNU, with or without AA-CW236 treatment to inactivate the MGMT protein. Error bars indicate mean ± SD; there were 4–6 replicates for each dose. If an asterisk is present over a point, that point is statistically different from the point with which it is vertically aligned (P
    Figure Legend Snippet: Dose-dependent responses of MEFs to MNU treatment with and without the MGMT inhibitor, AA-CW236. Panel A: effect of MNU and MNU plus AA-CW236 on MEF survival. Panel B: 6-TG r mutation frequency of MEFs with and without AA-CW236 using the gpt locus as the target for mutagenicity. Panel C: levels of m6G in the DNA of gpt delta MEFs treated with MNU, with or without AA-CW236 treatment to inactivate the MGMT protein. Error bars indicate mean ± SD; there were 4–6 replicates for each dose. If an asterisk is present over a point, that point is statistically different from the point with which it is vertically aligned (P

    Techniques Used: Mutagenesis

    Experimental system and workflow. Embryos from the gpt delta C57BL/6J mouse were converted into a MEF cell line by lentivirus-mediated transformation. MEFs were treated with MNU alone, or with MNU plus AA-CW236, an inhibitor of the MGMT repair protein. Cellular toxicity and mutagenicity were determined. Analytical measurement of m6G, a mutagenic DNA adduct of MNU, was performed at a dose of MNU that showed a strong mutagenic response (500 μM). Mutation distributions were determined in all possible 3-base contexts. The mutational patterns generated were then compared by cosine similarity with mutational signatures derived from sequencing of human tumors.
    Figure Legend Snippet: Experimental system and workflow. Embryos from the gpt delta C57BL/6J mouse were converted into a MEF cell line by lentivirus-mediated transformation. MEFs were treated with MNU alone, or with MNU plus AA-CW236, an inhibitor of the MGMT repair protein. Cellular toxicity and mutagenicity were determined. Analytical measurement of m6G, a mutagenic DNA adduct of MNU, was performed at a dose of MNU that showed a strong mutagenic response (500 μM). Mutation distributions were determined in all possible 3-base contexts. The mutational patterns generated were then compared by cosine similarity with mutational signatures derived from sequencing of human tumors.

    Techniques Used: Transformation Assay, Mutagenesis, Generated, Derivative Assay, Sequencing

    15) Product Images from "The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum"

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-016-0223-4

    Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment
    Figure Legend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment

    Techniques Used: Agarose Gel Electrophoresis, Isolation

    Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit
    Figure Legend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Formalin-fixed Paraffin-Embedded

    16) Product Images from "A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care"

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.15-0145

    Sensitivity of recombinase polymerase amplification–lateral flow (RPA-LF) to detect Leishmania infantum compared with real-time polymerase chain reaction (PCR) used as gold standard. Tenfold serial dilutions of L. infantum promastigotes in dog blood were extracted using Qiagen ® DNeasy blood and tissue kit and detected by real-time quantitative PCR (SYBRgreen) or RPA-LF. Parasite dilutions: 1 = 10 5 , 2 = 10 4 , 3 = 10 3 , 4 = 10 2 , 5 = 10, 6 = 1, and 7 = 0.1 parasites and Bl = uninfected dog blood. The top band is the control band; the lower band is the test band. This is a representative figure of two similar assays.
    Figure Legend Snippet: Sensitivity of recombinase polymerase amplification–lateral flow (RPA-LF) to detect Leishmania infantum compared with real-time polymerase chain reaction (PCR) used as gold standard. Tenfold serial dilutions of L. infantum promastigotes in dog blood were extracted using Qiagen ® DNeasy blood and tissue kit and detected by real-time quantitative PCR (SYBRgreen) or RPA-LF. Parasite dilutions: 1 = 10 5 , 2 = 10 4 , 3 = 10 3 , 4 = 10 2 , 5 = 10, 6 = 1, and 7 = 0.1 parasites and Bl = uninfected dog blood. The top band is the control band; the lower band is the test band. This is a representative figure of two similar assays.

    Techniques Used: Recombinase Polymerase Amplification, Flow Cytometry, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    17) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

    Techniques Used: DNA Methylation Assay, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, CpG Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    18) Product Images from "Development of real-time PCR for detection of Mycoplasma hominis"

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-35

    DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.
    Figure Legend Snippet: DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.

    Techniques Used: Polymerase Chain Reaction

    19) Product Images from "Creating conditional dual fluorescence labelled transgenic animals for studying function of small non-coding RNAs"

    Article Title: Creating conditional dual fluorescence labelled transgenic animals for studying function of small non-coding RNAs

    Journal: Connective tissue research

    doi: 10.1080/03008207.2016.1247834

    Dual fluorescence/ncRNA transgene construct and in vitro testing (A) MiR-365 flox transgenic construct structure. Mmu-miR-365-1 was cloned into transgene construct which adapted cre/loxP system to facilitate fluorescence switching from GFP to RFP. (B) Real-time PCR analysis of miR-365 level showed that miR-365 increased for about 25 fold in miR-365 flox construct and CMV-cre co-transfected NF1 cells than single plasmid transfection. For single transfection, 4 μg CMV-cre or miR-365 flox construct pCAG_G/R/M_miR-365 was transfected in six well plates with lipofectamine 2000. For double transfection, 3 μg of CMV-cre and 1 μg of pCAG_G/R/M_miR-365 was transfected. (C) Fluorescence signal showed that CMV-cre plasmid expressed no fluorescence, miR-365 flox construct pCAG_G/R/M_miR-365 expressed GFP and co-transfection of miR-365 flox construct and CMV-cre expressed RFP. Student t-test was used for statistics. (*) P
    Figure Legend Snippet: Dual fluorescence/ncRNA transgene construct and in vitro testing (A) MiR-365 flox transgenic construct structure. Mmu-miR-365-1 was cloned into transgene construct which adapted cre/loxP system to facilitate fluorescence switching from GFP to RFP. (B) Real-time PCR analysis of miR-365 level showed that miR-365 increased for about 25 fold in miR-365 flox construct and CMV-cre co-transfected NF1 cells than single plasmid transfection. For single transfection, 4 μg CMV-cre or miR-365 flox construct pCAG_G/R/M_miR-365 was transfected in six well plates with lipofectamine 2000. For double transfection, 3 μg of CMV-cre and 1 μg of pCAG_G/R/M_miR-365 was transfected. (C) Fluorescence signal showed that CMV-cre plasmid expressed no fluorescence, miR-365 flox construct pCAG_G/R/M_miR-365 expressed GFP and co-transfection of miR-365 flox construct and CMV-cre expressed RFP. Student t-test was used for statistics. (*) P

    Techniques Used: Fluorescence, Construct, In Vitro, Transgenic Assay, Clone Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cotransfection

    Characterization of transgene orientation (A) Three sets of primers were used to characterize the entire transgene (B) FloxP inserted in the same direction leads to loop out genes flanked by loxP cassetts as we designed. (C). FloxP inserted in the opposite direction leads to inversion of genes flanked by loxP cassetts. (D) The principle for designing primers used for checking orientation. For head-to-tail (HT) primers, a predicted PCR product will be detected when there is at least one head-to-tail orientation. In a similar way, head-to-head (HH) primers detect head to head orientations while tail-to-tail (TT) primers detect tail-to-tail orientations. (E) The three mice lines (17, 21, and 46) contain only head-to-tail junctions. In contrast, C29-1C, which had the high numbers of transgene copies, had head-to-tail, tail-to-tail and head-to-head junctions.
    Figure Legend Snippet: Characterization of transgene orientation (A) Three sets of primers were used to characterize the entire transgene (B) FloxP inserted in the same direction leads to loop out genes flanked by loxP cassetts as we designed. (C). FloxP inserted in the opposite direction leads to inversion of genes flanked by loxP cassetts. (D) The principle for designing primers used for checking orientation. For head-to-tail (HT) primers, a predicted PCR product will be detected when there is at least one head-to-tail orientation. In a similar way, head-to-head (HH) primers detect head to head orientations while tail-to-tail (TT) primers detect tail-to-tail orientations. (E) The three mice lines (17, 21, and 46) contain only head-to-tail junctions. In contrast, C29-1C, which had the high numbers of transgene copies, had head-to-tail, tail-to-tail and head-to-head junctions.

    Techniques Used: Polymerase Chain Reaction, Mouse Assay

    Identification of transgene genomic insertion site by inverse PCR (A) The steps for performing inverse PCR for identifying genomic location where transgene inserted. (B) Diagram illustration of genomic location of transgene insertion. The mouse genome blast results showed that the transgene insertion site on chromosome 4 is in an intergenic region between the gene encoding zinc finger protein 618, which is 308469 bp away and the gene encoding Regulator of G-protein signaling 3 isoform 3, which is 89334 bp away. The transgene insertion site is far away from the flanking genes on chromosome 4. (C) PCR verification of transgene insertion in miR-365 flox transgenic mice (TR) with forward primer recognizing genomic sequence and reverse primer recognizing transgene sequence. Four individual transgenic mice and four wild type mice genomic DNA were used for verification. Transgenic specific and non-specific bands were labelled.
    Figure Legend Snippet: Identification of transgene genomic insertion site by inverse PCR (A) The steps for performing inverse PCR for identifying genomic location where transgene inserted. (B) Diagram illustration of genomic location of transgene insertion. The mouse genome blast results showed that the transgene insertion site on chromosome 4 is in an intergenic region between the gene encoding zinc finger protein 618, which is 308469 bp away and the gene encoding Regulator of G-protein signaling 3 isoform 3, which is 89334 bp away. The transgene insertion site is far away from the flanking genes on chromosome 4. (C) PCR verification of transgene insertion in miR-365 flox transgenic mice (TR) with forward primer recognizing genomic sequence and reverse primer recognizing transgene sequence. Four individual transgenic mice and four wild type mice genomic DNA were used for verification. Transgenic specific and non-specific bands were labelled.

    Techniques Used: Inverse PCR, Polymerase Chain Reaction, Transgenic Assay, Mouse Assay, Sequencing

    20) Product Images from "Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion"

    Article Title: Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

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

    doi: 10.1073/pnas.1522223113

    PI3K, but not AKT, MAPKK, or SGK inhibition induces markers of DNA damage in BRCA1- mutant breast cancers. For A – C , HCC1937 cells were treated for 16 h with inhibitors as indicated. Immunoblotting of total cell lysates was performed with antibodies as indicated. ( A ) Induction of PAR and H2ax phosphorylation (γH2ax) following treatment with inhibitors of pan-PI3K (BKM120, 1.5 μM), PI3Kα (BYL719, 3 μM; PIK75, 0.5 μM), PI3Kβ (TGX221, 30 μM), the PARP-inhibitor Olaparib (5 μM), and inhibitors of AKT (MK2206, 1 μM), SGK (GSK650394, 10 μM), or MAPKK (GSK1120212, 5 nM). ( B ) Induction of PAR and γH2ax by the PIP3-mimetic PIT1. ( C ) Neither AKT nor SGK, nor the combination of AKT and SGK inhibitors, induces PAR or γH2ax. ( D and E ) Induction of DNA damage indicators in vivo. K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice were treated with two doses of drugs as indicated [BKM120 30 mg/kg by mouth (PO), BYL719 30 mg/kg PO, Olaparib 50 mg/kg i.p.] 8 and 2 h before killing. Tumors were immediately harvested and processed for immunoblotting of fresh tumor tissue lysates with antibodies as indicated ( D ) or fixed and stained for immunofluorescence with antibodies as indicated ( E ). Insets (400× magnification) show representative single cells stained for pATM ( Upper ) or pRPA ( Lower ).
    Figure Legend Snippet: PI3K, but not AKT, MAPKK, or SGK inhibition induces markers of DNA damage in BRCA1- mutant breast cancers. For A – C , HCC1937 cells were treated for 16 h with inhibitors as indicated. Immunoblotting of total cell lysates was performed with antibodies as indicated. ( A ) Induction of PAR and H2ax phosphorylation (γH2ax) following treatment with inhibitors of pan-PI3K (BKM120, 1.5 μM), PI3Kα (BYL719, 3 μM; PIK75, 0.5 μM), PI3Kβ (TGX221, 30 μM), the PARP-inhibitor Olaparib (5 μM), and inhibitors of AKT (MK2206, 1 μM), SGK (GSK650394, 10 μM), or MAPKK (GSK1120212, 5 nM). ( B ) Induction of PAR and γH2ax by the PIP3-mimetic PIT1. ( C ) Neither AKT nor SGK, nor the combination of AKT and SGK inhibitors, induces PAR or γH2ax. ( D and E ) Induction of DNA damage indicators in vivo. K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice were treated with two doses of drugs as indicated [BKM120 30 mg/kg by mouth (PO), BYL719 30 mg/kg PO, Olaparib 50 mg/kg i.p.] 8 and 2 h before killing. Tumors were immediately harvested and processed for immunoblotting of fresh tumor tissue lysates with antibodies as indicated ( D ) or fixed and stained for immunofluorescence with antibodies as indicated ( E ). Insets (400× magnification) show representative single cells stained for pATM ( Upper ) or pRPA ( Lower ).

    Techniques Used: Inhibition, Mutagenesis, In Vivo, Mouse Assay, Staining, Immunofluorescence

    Response to PI3K and PARP inhibition in vivo. Six different primary tumors from K14-Cre BRCA1 f/f p53 f/f females were propagated in vivo through syngeneic transplantation. Recipient females were randomized to treatments with BKM120 (30 mg⋅kg⋅ −1 d −1 by mouth), BYL719 (30 mg⋅kg⋅ −1 ⋅d −1 by mouth), Olaparib (50 mg⋅kg⋅ −1 d −1 i.p.), or their combination, and tumor volume was recorded every 2 d. Treatment endpoint was time to progression as defined by tumor volume doubling. ( A ) Two mice per treatment condition were killed after 8 and 72 h of drug treatments to assess biomarkers of response, as assessed with Ki67 and CC3 by IHC (magnification: 200×). These mice were given a last dose of drugs 2 h before killing. ( B ) Survival statistics (log-rank test) and Kaplan–Meier analysis of 132 recipient females randomized to treatments as indicated. ( C ) Resistance to PI3K inhibition. 18 FDG-PET-CT scan at baseline and after 48 h of administration of the PI3K inhibitor NVP-BKM120 (3 doses, 30 mg/kg PO) in sensitive and BKM120+Olaparib resistant K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice. The maximum standardized uptake value (SUVmax) of tumors before and after PI3K inhibitor administration is displayed in the bar graph. Tumors are marked with a yellow arrow. Two hours before killing, mice were injected with BrdU and given an additional dose of BKM120. Tumor sections were stained with anti-BrdU antibodies for immunofluoresence (red) (magnification: 200×) and mean fluorescence per cell of BrdU + cells was measured using volocity software. ( D ) A cell line was derived from the clinically resistant tumor and its PI3K/PARP-sensitive parental tumor. Cells were treated in vitro with drugs as indicated for 16 h, lysed, and blotted with antibodies as indicated.
    Figure Legend Snippet: Response to PI3K and PARP inhibition in vivo. Six different primary tumors from K14-Cre BRCA1 f/f p53 f/f females were propagated in vivo through syngeneic transplantation. Recipient females were randomized to treatments with BKM120 (30 mg⋅kg⋅ −1 d −1 by mouth), BYL719 (30 mg⋅kg⋅ −1 ⋅d −1 by mouth), Olaparib (50 mg⋅kg⋅ −1 d −1 i.p.), or their combination, and tumor volume was recorded every 2 d. Treatment endpoint was time to progression as defined by tumor volume doubling. ( A ) Two mice per treatment condition were killed after 8 and 72 h of drug treatments to assess biomarkers of response, as assessed with Ki67 and CC3 by IHC (magnification: 200×). These mice were given a last dose of drugs 2 h before killing. ( B ) Survival statistics (log-rank test) and Kaplan–Meier analysis of 132 recipient females randomized to treatments as indicated. ( C ) Resistance to PI3K inhibition. 18 FDG-PET-CT scan at baseline and after 48 h of administration of the PI3K inhibitor NVP-BKM120 (3 doses, 30 mg/kg PO) in sensitive and BKM120+Olaparib resistant K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice. The maximum standardized uptake value (SUVmax) of tumors before and after PI3K inhibitor administration is displayed in the bar graph. Tumors are marked with a yellow arrow. Two hours before killing, mice were injected with BrdU and given an additional dose of BKM120. Tumor sections were stained with anti-BrdU antibodies for immunofluoresence (red) (magnification: 200×) and mean fluorescence per cell of BrdU + cells was measured using volocity software. ( D ) A cell line was derived from the clinically resistant tumor and its PI3K/PARP-sensitive parental tumor. Cells were treated in vitro with drugs as indicated for 16 h, lysed, and blotted with antibodies as indicated.

    Techniques Used: Inhibition, In Vivo, Transplantation Assay, Mouse Assay, Immunohistochemistry, Positron Emission Tomography, Computed Tomography, Injection, Staining, Fluorescence, Software, Derivative Assay, In Vitro

    Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into cell biomass ( A ) or DNA ( B and C ) in response to PI3K and PARP inhibition. HCC1937 cells were cultured in the presence of 14 C 6 -glucose or 14 C 1 -glucose, BKM120 (1 μM) or Olaparib (5 μM), or their combination for 3 h as indicated. Scintillation was counted for the entire cell lysate ( A ); genomic DNA was extracted and 14 C measured ( B and C ). ( D ) Loss of PI3Kα leads to induction of H2AX phosphorylation (γH2AX) that can be rescued by nucleoside reconstitution. HCC1937 cells were depleted of PI3Kα by using siRNA for 48 h, and then treated with or without nucleosides for another 16 h. Cell lysates were subjected to immunoblotting with antibodies as indicated. ( E ) A radioactive label in the 6 position will measure flux through both oxidative and nonoxidative PPP, whereas a label in the 1 position will allow to measure flux through the nonoxidative PPP only.
    Figure Legend Snippet: Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into cell biomass ( A ) or DNA ( B and C ) in response to PI3K and PARP inhibition. HCC1937 cells were cultured in the presence of 14 C 6 -glucose or 14 C 1 -glucose, BKM120 (1 μM) or Olaparib (5 μM), or their combination for 3 h as indicated. Scintillation was counted for the entire cell lysate ( A ); genomic DNA was extracted and 14 C measured ( B and C ). ( D ) Loss of PI3Kα leads to induction of H2AX phosphorylation (γH2AX) that can be rescued by nucleoside reconstitution. HCC1937 cells were depleted of PI3Kα by using siRNA for 48 h, and then treated with or without nucleosides for another 16 h. Cell lysates were subjected to immunoblotting with antibodies as indicated. ( E ) A radioactive label in the 6 position will measure flux through both oxidative and nonoxidative PPP, whereas a label in the 1 position will allow to measure flux through the nonoxidative PPP only.

    Techniques Used: Derivative Assay, Inhibition, Cell Culture

    PI3K inhibition leads to decreased glycolytic flux through the nonoxidative pentose phosphate pathway. ( A ) Seahorse assay to determine overall glycolytic flux. Cells were seeded at 5,000 cells per well in a 24-well plate the night before the assay. Drugs were added at 3 h. ECAR was measured every minute for a total of 120 min in response to a glucose challenge, mitochondrial uncoupling to mobilize glycolytic reserve with oligomycin, and disruption of glycolysis with 2DG. Displayed is the ECAR over time of experimental quadruplicates ± SD. ( B ) Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into DNA in response to PI3K and PARP inhibition. Cells were treated with 1- 14 C- or 6- 14 C-glucose and drugs as indicated 8 h before lysis. Scintillation count was done on purified genomic DNA. Displayed is the 14 C-uptake of experimental triplicates ± SD, normalized to 14 C-uptake in the 6- 14 C-glucose control. ( C ) Effect of PI3K and AKT inhibition on Rib-phosphate synthesis in HCC1937 cells. Cells were treated with vehicle control, BKM120 (1 μM), or MK2206 (1 μM) for 3 h, followed by labeling with [U- 13 C 6 ]-glucose for 60 s and processed for mass spectrometry. ( D ) Nucleoside rescue of PI3K inhibitor-induced DNA damage indicators. Cells were treated for 16 h with drugs as indicated in the presence of a mixture of four nucleosides. ( E ) Determination of nucleotide levels in HCC1937 cells after 16 h of drug treatment with either BKM120 or MK2206 in the presence or absence of nucleosides. Determinations were done by competitive PCR, and displayed are the results of experimental triplicates. ( F ) Nucleoside rescue of PI3K inhibitor-induced decrease in DNA synthesis. Cells were treated for 8 h with drugs (BYL719, 2.5 μM; BKM120, 1 μM; or Olaparib, 5 μM) as indicated in the presence or absence of a mixture of four nucleosides. EdU was added in the last 2 h. Displayed are mean fluorescence of the EdU + population relative to vehicle control in experimental triplicates. Significance was P
    Figure Legend Snippet: PI3K inhibition leads to decreased glycolytic flux through the nonoxidative pentose phosphate pathway. ( A ) Seahorse assay to determine overall glycolytic flux. Cells were seeded at 5,000 cells per well in a 24-well plate the night before the assay. Drugs were added at 3 h. ECAR was measured every minute for a total of 120 min in response to a glucose challenge, mitochondrial uncoupling to mobilize glycolytic reserve with oligomycin, and disruption of glycolysis with 2DG. Displayed is the ECAR over time of experimental quadruplicates ± SD. ( B ) Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into DNA in response to PI3K and PARP inhibition. Cells were treated with 1- 14 C- or 6- 14 C-glucose and drugs as indicated 8 h before lysis. Scintillation count was done on purified genomic DNA. Displayed is the 14 C-uptake of experimental triplicates ± SD, normalized to 14 C-uptake in the 6- 14 C-glucose control. ( C ) Effect of PI3K and AKT inhibition on Rib-phosphate synthesis in HCC1937 cells. Cells were treated with vehicle control, BKM120 (1 μM), or MK2206 (1 μM) for 3 h, followed by labeling with [U- 13 C 6 ]-glucose for 60 s and processed for mass spectrometry. ( D ) Nucleoside rescue of PI3K inhibitor-induced DNA damage indicators. Cells were treated for 16 h with drugs as indicated in the presence of a mixture of four nucleosides. ( E ) Determination of nucleotide levels in HCC1937 cells after 16 h of drug treatment with either BKM120 or MK2206 in the presence or absence of nucleosides. Determinations were done by competitive PCR, and displayed are the results of experimental triplicates. ( F ) Nucleoside rescue of PI3K inhibitor-induced decrease in DNA synthesis. Cells were treated for 8 h with drugs (BYL719, 2.5 μM; BKM120, 1 μM; or Olaparib, 5 μM) as indicated in the presence or absence of a mixture of four nucleosides. EdU was added in the last 2 h. Displayed are mean fluorescence of the EdU + population relative to vehicle control in experimental triplicates. Significance was P

    Techniques Used: Inhibition, Derivative Assay, Lysis, Purification, Labeling, Mass Spectrometry, Polymerase Chain Reaction, DNA Synthesis, Fluorescence

    21) Product Images from "Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest"

    Article Title: Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest

    Journal: Genome Research

    doi: 10.1101/gr.201400.115

    DamIP-seq shows MAF1 recruitment at Pol III–bound genes. ( A ) Schematic representation of MAF1-DamK9A and EGFP-DamK9A chimeric constructs depicting the position of PCR primers. Two stable IMR90hTert clonal cell lines expressing each chimera (MAF1-A,
    Figure Legend Snippet: DamIP-seq shows MAF1 recruitment at Pol III–bound genes. ( A ) Schematic representation of MAF1-DamK9A and EGFP-DamK9A chimeric constructs depicting the position of PCR primers. Two stable IMR90hTert clonal cell lines expressing each chimera (MAF1-A,

    Techniques Used: Construct, Polymerase Chain Reaction, Expressing

    22) Product Images from "Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model"

    Article Title: Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model

    Journal: Theranostics

    doi: 10.7150/thno.26643

    PBMNCs are induced into neural stem cells by using Sendai virus. (A) A schematic representation of the neural stem cells induction procedure. PBMNCs: peripheral blood mononuclear cells. (B) Phase-contrast images of the PBMNCs, an iNSC clone, iNSCs in sphere and iNSCs in monolayer culture. Scale bars, 200 μm. (C) The growth curve of iNSCs P10, P20 and P30. (n=3 independent experiments). (D) Karyotype analysis of iNSCs. (E) Detection of remaining Sendai virus by PCR and immunocytochemistry staining. Scale bars, 50 μm. (F) Expression of transgenes ( SOX2 , OCT4 , KLF4 , cMYC ) and GAPDH reference gene at different passages of iNSCs. The values represent mean ± SEM in this figure and all the others in this article.
    Figure Legend Snippet: PBMNCs are induced into neural stem cells by using Sendai virus. (A) A schematic representation of the neural stem cells induction procedure. PBMNCs: peripheral blood mononuclear cells. (B) Phase-contrast images of the PBMNCs, an iNSC clone, iNSCs in sphere and iNSCs in monolayer culture. Scale bars, 200 μm. (C) The growth curve of iNSCs P10, P20 and P30. (n=3 independent experiments). (D) Karyotype analysis of iNSCs. (E) Detection of remaining Sendai virus by PCR and immunocytochemistry staining. Scale bars, 50 μm. (F) Expression of transgenes ( SOX2 , OCT4 , KLF4 , cMYC ) and GAPDH reference gene at different passages of iNSCs. The values represent mean ± SEM in this figure and all the others in this article.

    Techniques Used: Polymerase Chain Reaction, Immunocytochemistry, Staining, Expressing

    23) Product Images from "Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages"

    Article Title: Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102570

    NiCl 2 activates NLRP3 inflammasome pathway in BMDMs. ( A and B ) Relative mtROS amounts determined by MitoSOX-red staining of NiCl 2 -primed BMDMs. Scale bar 50 μm. ( C ) Relative cytosolic mtDNA expression in NiCl 2 -primed BMDMs. ( D ) NiCl 2 -induced changes in mitochondrial membrane potential (Ψm) in BMDMs measured by TMRM fluorescence. ( E ) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of NiCl 2 -treated BMDMs, and cleaved-caspase-1and cleaved- IL-1β in the supernatant. ( F ) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of Mito-TEMPO (500 μM)-pre-treated 1h before 24h of NiCl 2 stimulation. ( G ) Relative cytosolic mtDNA expression in NiCl 2 -treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. ( H ) Changes of mitochondrial membrane potential (Ψm) in NiCl 2 -treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. Data are presented with the means ± standard deviation (n=5). *p
    Figure Legend Snippet: NiCl 2 activates NLRP3 inflammasome pathway in BMDMs. ( A and B ) Relative mtROS amounts determined by MitoSOX-red staining of NiCl 2 -primed BMDMs. Scale bar 50 μm. ( C ) Relative cytosolic mtDNA expression in NiCl 2 -primed BMDMs. ( D ) NiCl 2 -induced changes in mitochondrial membrane potential (Ψm) in BMDMs measured by TMRM fluorescence. ( E ) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of NiCl 2 -treated BMDMs, and cleaved-caspase-1and cleaved- IL-1β in the supernatant. ( F ) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of Mito-TEMPO (500 μM)-pre-treated 1h before 24h of NiCl 2 stimulation. ( G ) Relative cytosolic mtDNA expression in NiCl 2 -treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. ( H ) Changes of mitochondrial membrane potential (Ψm) in NiCl 2 -treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. Data are presented with the means ± standard deviation (n=5). *p

    Techniques Used: Staining, Expressing, Fluorescence, Standard Deviation

    24) Product Images from "Function and Evolution of DNA Methylation in Nasonia vitripennis"

    Article Title: Function and Evolution of DNA Methylation in Nasonia vitripennis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003872

    DNA methylation and gene conservation. (A) Phylogenetic tree of eight insect species: Nasonia vitripennis , Apis mellifera , Tribolium castaneum , Bombyx mori , Anopheles gambiae , Drosophila melanogaster , Pediculus humanus and Acyrthosiphon pisum . The methylation status and correlating factors were plotted in (B–F) for four groups of genes: all 5,039 Nasonia single-copy genes with one or zero ortholog in seven other insect species, 2,374 genes with one orthologs in all eight insect species, 443 genes with one orthologs in Apis and Nasonia but missing in other six species, and 320 genes present only in Nasonia . The y -axes plotted in (B–F) are (B): proportion of methylated (blue) and non-methylated genes (red); (C): percentage of methylated CpG sites in methylated genes; (D): adult RNA-seq expression levels (log 10 FPKM); (E): coefficient of variation of expression level in tiling array across six developmental stages; (F): number of expressed tissues. (G) Top: Phylogenetic tree of three Nasonia species: N. longicornis (L), N. giraulti (G) and N. vitripennis (V). Bottom: boxplots of nucleotide substitution rates between V–L, V–G and L–G.
    Figure Legend Snippet: DNA methylation and gene conservation. (A) Phylogenetic tree of eight insect species: Nasonia vitripennis , Apis mellifera , Tribolium castaneum , Bombyx mori , Anopheles gambiae , Drosophila melanogaster , Pediculus humanus and Acyrthosiphon pisum . The methylation status and correlating factors were plotted in (B–F) for four groups of genes: all 5,039 Nasonia single-copy genes with one or zero ortholog in seven other insect species, 2,374 genes with one orthologs in all eight insect species, 443 genes with one orthologs in Apis and Nasonia but missing in other six species, and 320 genes present only in Nasonia . The y -axes plotted in (B–F) are (B): proportion of methylated (blue) and non-methylated genes (red); (C): percentage of methylated CpG sites in methylated genes; (D): adult RNA-seq expression levels (log 10 FPKM); (E): coefficient of variation of expression level in tiling array across six developmental stages; (F): number of expressed tissues. (G) Top: Phylogenetic tree of three Nasonia species: N. longicornis (L), N. giraulti (G) and N. vitripennis (V). Bottom: boxplots of nucleotide substitution rates between V–L, V–G and L–G.

    Techniques Used: DNA Methylation Assay, Methylation, RNA Sequencing Assay, Expressing

    25) Product Images from "Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding"

    Article Title: Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1977

    DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.
    Figure Legend Snippet: DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.

    Techniques Used: Infection, Clone Assay

    Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.
    Figure Legend Snippet: Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.

    Techniques Used: Plasmid Preparation, Mouse Assay, Transplantation Assay, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing

    26) Product Images from "Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding"

    Article Title: Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1977

    DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.
    Figure Legend Snippet: DNA barcode library and delivery. (a) Histogram displaying barcode copy numbers from a lentiviral library. Additional lentiviral libraries are shown in Supplementary Fig. 1 , together with the negative controls to demonstrate the level of background noise for this experiment. (b) Histogram showing the number of barcode(s) that each HSC clone receives after infection. 95 HSC clones were examined in total. This distribution fits a normal distribution shown in Supplementary Fig. 3 . (c) Monte Carlo simulation of the null hypothesis that more than 95% of the barcodes represent single cells. The P value is plotted against the size of the cell population whose barcodes are recovered in the result.

    Techniques Used: Infection, Clone Assay

    Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.
    Figure Legend Snippet: Experimental workflow. A DNA barcode consists of a common 6bp library ID at the 5′ end followed by a random 27bp cellular barcode. In the figure, different colors represent different barcode sequences. A lentiviral vector delivers a large library of barcodes into a small number of cells such that each cell receives a unique barcode. Barcodes replicate with the cells in the recipient mice after transplantation. Afterwards, the progeny of the donor cells are harvested. Barcodes are recovered from the genomic DNA using PCR and analyzed using high throughput sequencing (Illumina GA II). The 6bp library ID helps to identify barcodes in the sequencing result. Identical 33bp barcodes are combined allowing for mismatches and indels up to 2bp in total. The barcodes are then compared across different cell populations that originate from the same starting cell population.

    Techniques Used: Plasmid Preparation, Mouse Assay, Transplantation Assay, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing

    27) Product Images from "Enhancement of ascomycin production via a combination of atmospheric and room temperature plasma mutagenesis in Streptomyces hygroscopicus and medium optimization"

    Article Title: Enhancement of ascomycin production via a combination of atmospheric and room temperature plasma mutagenesis in Streptomyces hygroscopicus and medium optimization

    Journal: AMB Express

    doi: 10.1186/s13568-019-0749-x

    Transcriptional analysis of ascomycin biosynthetic gene cluster in S. hygroscopicus . a Genetic organization of co-transcription units in ascomycin biosynthetic gene cluster. b Co-transcriptional analysis of the ascomycin biosynthetic gene cluster by RT-PCR. Genomic DNA (gDNA) and cDNA of S. hygroscopicus 14891 strain were used for PCR amplification. c Relative expression levels of ascomycin biosynthetic gene cluster in S. hygroscopicus SFK-36, compared with those in ATCC 14891 strain. Totally, seven genes were selected to indicate the expression level of co-transcription units (***P
    Figure Legend Snippet: Transcriptional analysis of ascomycin biosynthetic gene cluster in S. hygroscopicus . a Genetic organization of co-transcription units in ascomycin biosynthetic gene cluster. b Co-transcriptional analysis of the ascomycin biosynthetic gene cluster by RT-PCR. Genomic DNA (gDNA) and cDNA of S. hygroscopicus 14891 strain were used for PCR amplification. c Relative expression levels of ascomycin biosynthetic gene cluster in S. hygroscopicus SFK-36, compared with those in ATCC 14891 strain. Totally, seven genes were selected to indicate the expression level of co-transcription units (***P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Expressing

    28) Product Images from "Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells"

    Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells

    Journal: eLife

    doi: 10.7554/eLife.36187

    GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.
    Figure Legend Snippet: GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.

    Techniques Used: FACS, Mouse Assay, RNA Sequencing Assay, Sequencing, Expressing, Purification

    29) Product Images from "Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs"

    Article Title: Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00701

    Frequencies and levels of sequencing reads for the SLA class I alleles. The identified SLA class I alleles at each of the three classical SLA class I loci in the four different pig populations are shown; (A) Göttingen minipigs ( N = 19), (B) Kenyan pigs ( N = 9), (C) Danish pigs ( N = 13), and (D) Belgian pigs ( N = 29). For each allele, the frequency of animals having the allele (light gray bars) and level of sequencing reads (dark gray bars) are shown here as median value with error bars indicating range of transcription. The SLA-1*an02 allele could not be verified by the PCR-SSP method.
    Figure Legend Snippet: Frequencies and levels of sequencing reads for the SLA class I alleles. The identified SLA class I alleles at each of the three classical SLA class I loci in the four different pig populations are shown; (A) Göttingen minipigs ( N = 19), (B) Kenyan pigs ( N = 9), (C) Danish pigs ( N = 13), and (D) Belgian pigs ( N = 29). For each allele, the frequency of animals having the allele (light gray bars) and level of sequencing reads (dark gray bars) are shown here as median value with error bars indicating range of transcription. The SLA-1*an02 allele could not be verified by the PCR-SSP method.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    30) Product Images from "Characterization of Cytosine Methylation and the DNA Methyltransferases of Toxoplasma gondii"

    Article Title: Characterization of Cytosine Methylation and the DNA Methyltransferases of Toxoplasma gondii

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.18644

    Verification of the cytosine methylation sites in the T. gondii genome using enzymes ( HpaII/MspI ) coupled with PCR. Hpa II and Msp I were used to digest gDNA followed by PCR or qPCR. A: At the T-IV-1336110+ site, where partial methylation was presented in tachyzoites, the methylation-sensitive enzyme Hpa II did not fully cut the gDNA at this site, and PCR product appeared; however, methylation-insensitive enzyme Msp I cut the gDNA at this site thoroughly, and no PCR products were obtained. B: qPCR validation was conducted after the gDNA was digested by Hpa II and Msp I, respectively. All results were obtained from three repetitive experiments with three replicates. T -tachyzoites, B -bradyzoites. Error bars: SEM.
    Figure Legend Snippet: Verification of the cytosine methylation sites in the T. gondii genome using enzymes ( HpaII/MspI ) coupled with PCR. Hpa II and Msp I were used to digest gDNA followed by PCR or qPCR. A: At the T-IV-1336110+ site, where partial methylation was presented in tachyzoites, the methylation-sensitive enzyme Hpa II did not fully cut the gDNA at this site, and PCR product appeared; however, methylation-insensitive enzyme Msp I cut the gDNA at this site thoroughly, and no PCR products were obtained. B: qPCR validation was conducted after the gDNA was digested by Hpa II and Msp I, respectively. All results were obtained from three repetitive experiments with three replicates. T -tachyzoites, B -bradyzoites. Error bars: SEM.

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

    The relationship between DNA methylation and gene expression. 190 genes with different mCpG levels in the upstream region, and 576 genes with different mC levels in the CDS region, and all with a transcription level of 1" were included in the analysis. Among these 190 genes, the proportion of significantly higher methylation (bradyzoite/tachyzoites≥10) is slightly higher (approximately 1.15 times higher) in the down-regulated genes than in the up-regulated genes of bradyzoites, when compared to tachyzoite gene transcription; but among the 576 genes, the proportion of significantly higher methylation (bradyzoite/tachyzoites≥10) is apparently higher in the up-regulated genes than in the down-regulated genes of bradyzoites, when compared to tachyzoite gene transcription. " title="... down-regulated genes than in the up-regulated genes of bradyzoites, when compared to tachyzoite gene transcription; but among ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: The relationship between DNA methylation and gene expression. 190 genes with different mCpG levels in the upstream region, and 576 genes with different mC levels in the CDS region, and all with a transcription level of "|log 2 expression ration| > 1" were included in the analysis. Among these 190 genes, the proportion of significantly higher methylation (bradyzoite/tachyzoites≥10) is slightly higher (approximately 1.15 times higher) in the down-regulated genes than in the up-regulated genes of bradyzoites, when compared to tachyzoite gene transcription; but among the 576 genes, the proportion of significantly higher methylation (bradyzoite/tachyzoites≥10) is apparently higher in the up-regulated genes than in the down-regulated genes of bradyzoites, when compared to tachyzoite gene transcription.

    Techniques Used: DNA Methylation Assay, Expressing, Methylation

    Detection of Tgdnmta and Tgdnmtb transcription in tachyzoites and bradyzoites. Each qPCR reaction were performed in triplicate, and the detection were repeated for three times. Relative transcription levels of Tgdnmta and Tgdnmtb genes were normalized to the transcription level of housekeeping gene GAPDH and were calculated using the 2 -ΔΔCt method. The differences of Tgdnmt transcriptional levels between tachyzoites and bradyzoites were analyzed with an independent t-test in SPSS13.0 software (Chicago, IL, USA). The transcription levels of both Tgdnmta and Tgdnmtb in bradyzoites were significantly higher than those in tachyzoites, and especially for Tgdnmtb , the relative transcriptional level in bradyzoites was approximately 300 fold higher than that of the transcription level found in tachyzoites. Error bars: SEM.
    Figure Legend Snippet: Detection of Tgdnmta and Tgdnmtb transcription in tachyzoites and bradyzoites. Each qPCR reaction were performed in triplicate, and the detection were repeated for three times. Relative transcription levels of Tgdnmta and Tgdnmtb genes were normalized to the transcription level of housekeeping gene GAPDH and were calculated using the 2 -ΔΔCt method. The differences of Tgdnmt transcriptional levels between tachyzoites and bradyzoites were analyzed with an independent t-test in SPSS13.0 software (Chicago, IL, USA). The transcription levels of both Tgdnmta and Tgdnmtb in bradyzoites were significantly higher than those in tachyzoites, and especially for Tgdnmtb , the relative transcriptional level in bradyzoites was approximately 300 fold higher than that of the transcription level found in tachyzoites. Error bars: SEM.

    Techniques Used: Real-time Polymerase Chain Reaction, Software

    Comparison of global DNA methylation in tachyzoite and bradyzoite genomes . A-B: Methylation context distribution of m 5 C in tachyzoites and bradyzoites; H represents any nucleotide A, T, and C. C: comparison of the proportion of methylated cytosines within the compartment of the genes.
    Figure Legend Snippet: Comparison of global DNA methylation in tachyzoite and bradyzoite genomes . A-B: Methylation context distribution of m 5 C in tachyzoites and bradyzoites; H represents any nucleotide A, T, and C. C: comparison of the proportion of methylated cytosines within the compartment of the genes.

    Techniques Used: DNA Methylation Assay, Methylation

    31) Product Images from "Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy"

    Article Title: Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

    Journal: Autophagy

    doi: 10.4161/15548627.2014.981915

    (See previous page.) Mitochondrial DNA release following infection and requirement for mitochondria for inflammasome activation by P. aeruginosa . ( A ) and ( B ) , qPCR analysis of cytosolic mitochondrial DNA (mtDNA) relative to nuclear DNA in macrophages pretreated ( A ) with Mito-TEMPO (500 μM) or 3-MA (10 mM) or control or Lc3b siRNA ( B ) and infected with PA103ΔUΔT (MOI 25) for 4 h or uninfected (Basal) as shown. Columns show means of 3 independent determinations; error bars are SEM. ( C ) Mitochondrial content of J774A.1 cells exposed to ethidium bromide (EtBr) at the indicated concentration (ng/ml) measured by qPCR (normalized to untreated cells; upper panel) and immunoblot for the mitochondrial protein ATPIF1 (lower panel) at low and high exposure time; TUBB5 is shown as a loading control. ( D ) Mitochondrial content of control or ethidium bromide-treated J774A.1 cells (ρJ774A.1) assayed by flow cytometry of MitoTracker Green stained cells. ( E ) Flow cytometry of J774A.1 and ρ°J774A.1 cells left uninfected (Basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and stained with MitoSOX Red. ( F ) J774A.1 cells grown in the absence or presence of 500 ng/ml ethidium bromide (EtBr) were left untreated (basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and analyzed as described in Figure 1A . *** indicates significant differences between the levels in the presence and absence of the EtBr (500 ng/ml), P
    Figure Legend Snippet: (See previous page.) Mitochondrial DNA release following infection and requirement for mitochondria for inflammasome activation by P. aeruginosa . ( A ) and ( B ) , qPCR analysis of cytosolic mitochondrial DNA (mtDNA) relative to nuclear DNA in macrophages pretreated ( A ) with Mito-TEMPO (500 μM) or 3-MA (10 mM) or control or Lc3b siRNA ( B ) and infected with PA103ΔUΔT (MOI 25) for 4 h or uninfected (Basal) as shown. Columns show means of 3 independent determinations; error bars are SEM. ( C ) Mitochondrial content of J774A.1 cells exposed to ethidium bromide (EtBr) at the indicated concentration (ng/ml) measured by qPCR (normalized to untreated cells; upper panel) and immunoblot for the mitochondrial protein ATPIF1 (lower panel) at low and high exposure time; TUBB5 is shown as a loading control. ( D ) Mitochondrial content of control or ethidium bromide-treated J774A.1 cells (ρJ774A.1) assayed by flow cytometry of MitoTracker Green stained cells. ( E ) Flow cytometry of J774A.1 and ρ°J774A.1 cells left uninfected (Basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and stained with MitoSOX Red. ( F ) J774A.1 cells grown in the absence or presence of 500 ng/ml ethidium bromide (EtBr) were left untreated (basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and analyzed as described in Figure 1A . *** indicates significant differences between the levels in the presence and absence of the EtBr (500 ng/ml), P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Infection, Activation Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Flow Cytometry, Cytometry, Staining

    (See previous page.) Mitochondrial DNA activates the NLRC4 inflammasome independently of Aim2 . ( A ) BMDMs were transfected with 3 μg DNASE1, lactate dehydrogenase (LDH), or heat-inactivated (HI) DNASE1 as shown and then infected with PA103ΔUΔT (MOI 25) for 4 h. The panels show immunoblot of the indicated proteins and TUBB5 as a loading control as in Figure 1A , levels of IL1B and TNF as in Figure 1A and qPCR analysis of cytosolic mtDNA as in Figure 4A . *** indicates significant difference from HI DNASE1, P
    Figure Legend Snippet: (See previous page.) Mitochondrial DNA activates the NLRC4 inflammasome independently of Aim2 . ( A ) BMDMs were transfected with 3 μg DNASE1, lactate dehydrogenase (LDH), or heat-inactivated (HI) DNASE1 as shown and then infected with PA103ΔUΔT (MOI 25) for 4 h. The panels show immunoblot of the indicated proteins and TUBB5 as a loading control as in Figure 1A , levels of IL1B and TNF as in Figure 1A and qPCR analysis of cytosolic mtDNA as in Figure 4A . *** indicates significant difference from HI DNASE1, P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Transfection, Infection, Real-time Polymerase Chain Reaction

    32) Product Images from "Genomic profiling of murine mammary tumors identifies potential personalized drug targets for p53-deficient mammary cancers"

    Article Title: Genomic profiling of murine mammary tumors identifies potential personalized drug targets for p53-deficient mammary cancers

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.025239

    Murine Trp53 -null tumor datasets. Sequencing and microarray technologies were used to produce four Trp53 -null tumor datasets of varying sizes: (i) whole genome sequencing ( n =12), (ii) exome sequencing ( n =25), (iii) DNA copy-number microarray ( n =43) and (iv) gene expression microarray ( n =43). The intrinsic class of each sample is displayed on the dendrogram, with colored boxes being previously identified human subtype counterparts ( Pfefferle et al., 2013 ). The hierarchical clustering location of each p53-null tumor within the datasets is displayed as a vertical black strip. *The Trp53 -null transplant model produces heterogeneous tumors that primarily develop into one of these three murine expression subtypes. For each dataset, the number of tumors studied from each of the three murine classes highlighted by ‘*’ is displayed on the right-hand side of the figure.
    Figure Legend Snippet: Murine Trp53 -null tumor datasets. Sequencing and microarray technologies were used to produce four Trp53 -null tumor datasets of varying sizes: (i) whole genome sequencing ( n =12), (ii) exome sequencing ( n =25), (iii) DNA copy-number microarray ( n =43) and (iv) gene expression microarray ( n =43). The intrinsic class of each sample is displayed on the dendrogram, with colored boxes being previously identified human subtype counterparts ( Pfefferle et al., 2013 ). The hierarchical clustering location of each p53-null tumor within the datasets is displayed as a vertical black strip. *The Trp53 -null transplant model produces heterogeneous tumors that primarily develop into one of these three murine expression subtypes. For each dataset, the number of tumors studied from each of the three murine classes highlighted by ‘*’ is displayed on the right-hand side of the figure.

    Techniques Used: Sequencing, Microarray, Expressing, Stripping Membranes

    Human counterparts of Trp53 -null transplant tumors. (A) Genes highly expressed within each Trp53 -null transplant class were identified using a two-class (class x versus all others) SAM analysis (FDR 0%) across our 385-sample murine microarray dataset. The standardized average of these gene signatures was calculated across more than 3000 human tumors and displayed by intrinsic subtype. (B) Tumor differentiation scores (D-Scores) ( Prat et al., 2010 ) were calculated for all 385 murine samples and displayed by intrinsic class. The D-Scores of the three Trp53 -null transplant classes were compared using a Student's t -test.
    Figure Legend Snippet: Human counterparts of Trp53 -null transplant tumors. (A) Genes highly expressed within each Trp53 -null transplant class were identified using a two-class (class x versus all others) SAM analysis (FDR 0%) across our 385-sample murine microarray dataset. The standardized average of these gene signatures was calculated across more than 3000 human tumors and displayed by intrinsic subtype. (B) Tumor differentiation scores (D-Scores) ( Prat et al., 2010 ) were calculated for all 385 murine samples and displayed by intrinsic class. The D-Scores of the three Trp53 -null transplant classes were compared using a Student's t -test.

    Techniques Used: Microarray

    DNA copy-number analysis. Displayed in genomic order are the median class DNA copy-number levels for (A) p53null-Basal Ex , (B) p53null-Claudin-low Ex and (C) p53null-Luminal Ex tumors. DNA copy-number changes enriched within each of the three Trp53 -null transplant classes were identified using a two-class (class x versus all others) SAM analysis. Genomic regions of significant gain are labeled in red and regions of significant loss are labeled in green.
    Figure Legend Snippet: DNA copy-number analysis. Displayed in genomic order are the median class DNA copy-number levels for (A) p53null-Basal Ex , (B) p53null-Claudin-low Ex and (C) p53null-Luminal Ex tumors. DNA copy-number changes enriched within each of the three Trp53 -null transplant classes were identified using a two-class (class x versus all others) SAM analysis. Genomic regions of significant gain are labeled in red and regions of significant loss are labeled in green.

    Techniques Used: Labeling

    33) Product Images from "Comparative Genomic Analysis of Asian Haemorrhagic Septicaemia-Associated Strains of Pasteurella multocida Identifies More than 90 Haemorrhagic Septicaemia-Specific Genes"

    Article Title: Comparative Genomic Analysis of Asian Haemorrhagic Septicaemia-Associated Strains of Pasteurella multocida Identifies More than 90 Haemorrhagic Septicaemia-Specific Genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130296

    Unrooted neighbour-joining trees showing the phylogenetic relationship between various strains. A. Relationship between Gallibacterium anatis , Mannheimia haemolytica , Pasteurella dagmatis , Pasteurella bettyae and the P . multocida strains Pm70, 36950, HN06, P3480, X73, VP161, Anand1C, Anand1B, Anand1P, Anand1G, P1059, P52VAC, VTCCBAA264, M1404 and the twelve Pakistani and Thai isolates. B. Relationship between the P . multocida strains. C. Relationship between the HS-associated P . multocida B:2 strain M1404 and the twelve Pakistani and Thai isolates. Phylogenetic relatedness for all comparisons was determined by analysis of only the single nucleotide polymorphisms found at conserved positions in all genomes of the comparison set (CG-SNPs); 789 shared positions for the tree in panel A, 7,829 shared positions for the tree in panel B and 722 shared positions for the tree in panel C. Trees were rendered with SplitsTree v4.11.3 [ 34 ]. The line segments above the trees with the number '0.01' indicate the branch length representing a genetic change of 0.01.
    Figure Legend Snippet: Unrooted neighbour-joining trees showing the phylogenetic relationship between various strains. A. Relationship between Gallibacterium anatis , Mannheimia haemolytica , Pasteurella dagmatis , Pasteurella bettyae and the P . multocida strains Pm70, 36950, HN06, P3480, X73, VP161, Anand1C, Anand1B, Anand1P, Anand1G, P1059, P52VAC, VTCCBAA264, M1404 and the twelve Pakistani and Thai isolates. B. Relationship between the P . multocida strains. C. Relationship between the HS-associated P . multocida B:2 strain M1404 and the twelve Pakistani and Thai isolates. Phylogenetic relatedness for all comparisons was determined by analysis of only the single nucleotide polymorphisms found at conserved positions in all genomes of the comparison set (CG-SNPs); 789 shared positions for the tree in panel A, 7,829 shared positions for the tree in panel B and 722 shared positions for the tree in panel C. Trees were rendered with SplitsTree v4.11.3 [ 34 ]. The line segments above the trees with the number '0.01' indicate the branch length representing a genetic change of 0.01.

    Techniques Used:

    34) Product Images from "Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells"

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1152

    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P
    Figure Legend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    ( A ) Heatmap showing GRO-Seq, Pol2-S2P, γH2AX ChIP-Seq, and 8-oxodG OxiDIP-Seq signals at the gene body, and within the 5 kb both upstream the TSS and downstream the TTS of the RefSeq genes. Gene clusters identified by SeqMINER unbiased k-means clustering (Cluster #1–#4), and the number of genes contained within each cluster, as indicated. ( B ) Read density profiles of 8-oxodG OxiDIP-Seq (red) and γH2AX ChIP-Seq (black) in Cluster #1–#4, as indicated. ( C ) Box plot showing the distribution of transcription levels (RPKM, measured by GRO-seq) of human genes within each cluster in MCF10A cells. ( D ) Box plot showing the length distribution of human genes within each cluster in MCF10A cells ( P
    Figure Legend Snippet: ( A ) Heatmap showing GRO-Seq, Pol2-S2P, γH2AX ChIP-Seq, and 8-oxodG OxiDIP-Seq signals at the gene body, and within the 5 kb both upstream the TSS and downstream the TTS of the RefSeq genes. Gene clusters identified by SeqMINER unbiased k-means clustering (Cluster #1–#4), and the number of genes contained within each cluster, as indicated. ( B ) Read density profiles of 8-oxodG OxiDIP-Seq (red) and γH2AX ChIP-Seq (black) in Cluster #1–#4, as indicated. ( C ) Box plot showing the distribution of transcription levels (RPKM, measured by GRO-seq) of human genes within each cluster in MCF10A cells. ( D ) Box plot showing the length distribution of human genes within each cluster in MCF10A cells ( P

    Techniques Used: Chromatin Immunoprecipitation

    35) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Affinity Magnetic Separation, Incubation, Mouse Assay, Sonication, Generated, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

    Techniques Used: DNA Methylation Assay, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, CpG Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    36) Product Images from "Folate deficiency exacerbates apoptosis by inducing hypomethylation and resultant overexpression of DR4 together with altering DNMTs in Alzheimer’s disease"

    Article Title: Folate deficiency exacerbates apoptosis by inducing hypomethylation and resultant overexpression of DR4 together with altering DNMTs in Alzheimer’s disease

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    Cell viability examination by MTT assay in SH-SY5Y cells, which was administrated various concentration of folate for 144 h (A) and folate 0 ug/ml for various times (B). * P
    Figure Legend Snippet: Cell viability examination by MTT assay in SH-SY5Y cells, which was administrated various concentration of folate for 144 h (A) and folate 0 ug/ml for various times (B). * P

    Techniques Used: MTT Assay, Concentration Assay

    37) Product Images from "Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation"

    Article Title: Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI84262

    Melanocytes isolated from TSC patients’ hypomelanotic macules accumulate early-stage melanosomes.
    Figure Legend Snippet: Melanocytes isolated from TSC patients’ hypomelanotic macules accumulate early-stage melanosomes.

    Techniques Used: Isolation

    mTORC1 is hyperactive in melanocytes isolated from TSC patients’ hypomelanotic macules.
    Figure Legend Snippet: mTORC1 is hyperactive in melanocytes isolated from TSC patients’ hypomelanotic macules.

    Techniques Used: Isolation

    38) Product Images from "Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer"

    Article Title: Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-17-0593

    Analytical process and characteristics of the cohort of cases of acquired resistance to immune checkpoint inhibitors (A) Schematic representation of the repeat biopsy program and sample analysis. Tumor specimens (and corresponding PDXs when available) collected at the time of resistance to ICIs and before treatment with immune checkpoint inhibitors along with germline DNA were analyzed using whole exome sequencing. For select samples with sufficient material, RNA sequencing and quantitative immunofluorescence were also performed. (B) Pie-chart illustrating the types of therapies received by patients in this study. (C) Swimmer’s plot indicating time of response, resistance to ICIs, and length of time on therapy for individual patients.
    Figure Legend Snippet: Analytical process and characteristics of the cohort of cases of acquired resistance to immune checkpoint inhibitors (A) Schematic representation of the repeat biopsy program and sample analysis. Tumor specimens (and corresponding PDXs when available) collected at the time of resistance to ICIs and before treatment with immune checkpoint inhibitors along with germline DNA were analyzed using whole exome sequencing. For select samples with sufficient material, RNA sequencing and quantitative immunofluorescence were also performed. (B) Pie-chart illustrating the types of therapies received by patients in this study. (C) Swimmer’s plot indicating time of response, resistance to ICIs, and length of time on therapy for individual patients.

    Techniques Used: Sequencing, RNA Sequencing Assay, Immunofluorescence

    39) Product Images from "HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site"

    Article Title: HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.01135-18

    Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.
    Figure Legend Snippet: Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.

    Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification, Expressing, Clone Assay, Variant Assay

    Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.
    Figure Legend Snippet: Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.

    Techniques Used: Inhibition, Produced, Expressing, Activity Assay

    Development of HIV-1 resistance to Cas9/sgPBS3. Viruses at the peaks of replication in Cas9/sgPBS3-expressing SupT1 cells were collected and sequenced. Indels in the PBS DNA were detected and are shown for the first, second, and third rounds of replication. The frequency of each indel in the sequenced DNA clones is also presented.
    Figure Legend Snippet: Development of HIV-1 resistance to Cas9/sgPBS3. Viruses at the peaks of replication in Cas9/sgPBS3-expressing SupT1 cells were collected and sequenced. Indels in the PBS DNA were detected and are shown for the first, second, and third rounds of replication. The frequency of each indel in the sequenced DNA clones is also presented.

    Techniques Used: Expressing, Clone Assay

    The PBS-M1 and PBS-M2 mutations allow HIV-1 to resist Cas9/sgPBS1. (A) Depiction of the M1 and M2 mutations (in red letters) in the PBS. (B) M1 and M2 mutations resist Cas9/sgPBS1 inhibition in short-term infection. Wild-type HIV-1 or the M1 or M2 mutant, with the same p24 amounts, was used to infect SupT1 cells expressing the Cas9 clone (control) or Cas9/sgPBS1. Forty hours after infection, the levels of HIV-1 in the culture supernatants were determined by infecting the TZM-bl indicator cells. Results shown are the average values from three independent infection experiments. Levels of each virus from the control cells are set at 100. ***, P
    Figure Legend Snippet: The PBS-M1 and PBS-M2 mutations allow HIV-1 to resist Cas9/sgPBS1. (A) Depiction of the M1 and M2 mutations (in red letters) in the PBS. (B) M1 and M2 mutations resist Cas9/sgPBS1 inhibition in short-term infection. Wild-type HIV-1 or the M1 or M2 mutant, with the same p24 amounts, was used to infect SupT1 cells expressing the Cas9 clone (control) or Cas9/sgPBS1. Forty hours after infection, the levels of HIV-1 in the culture supernatants were determined by infecting the TZM-bl indicator cells. Results shown are the average values from three independent infection experiments. Levels of each virus from the control cells are set at 100. ***, P

    Techniques Used: Inhibition, Infection, Mutagenesis, Expressing

    Illustration of the effect of PAM position in the PBS DNA on Cas9/sgRNA restriction of HIV-1 infection. If PAM is positioned on the plus strand of the PBS, HIV-1 is able to escape by acquiring and maintaining single- or double-nucleotide mutations in the PBS. If PAM is located on the minus strand of the PBS DNA, Cas9/sgRNA is able to target and cleave viral cDNA that is reverse transcribed from viral RNA that bears mutated PBS sequences. Based on these mechanisms, it can be predicted that a greater and more persistent suppression of HIV-1 infection can be achieved with a Cas9 variant using the CCA PAM that is on the minus strand of the PBS DNA and also allows the design of sgRNA with its seed region (14 to 15 nt) included in the PBS.
    Figure Legend Snippet: Illustration of the effect of PAM position in the PBS DNA on Cas9/sgRNA restriction of HIV-1 infection. If PAM is positioned on the plus strand of the PBS, HIV-1 is able to escape by acquiring and maintaining single- or double-nucleotide mutations in the PBS. If PAM is located on the minus strand of the PBS DNA, Cas9/sgRNA is able to target and cleave viral cDNA that is reverse transcribed from viral RNA that bears mutated PBS sequences. Based on these mechanisms, it can be predicted that a greater and more persistent suppression of HIV-1 infection can be achieved with a Cas9 variant using the CCA PAM that is on the minus strand of the PBS DNA and also allows the design of sgRNA with its seed region (14 to 15 nt) included in the PBS.

    Techniques Used: Infection, Variant Assay

    Development of HIV-1 resistance to Cas9/sgPBS1. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS1-expressing SupT1 cells were harvested. Viral RNA was extracted and subjected to RT-PCR and sequencing. Each indel in the PBS was registered, and its frequency in the sequenced DNA clones was calculated. The bar graph shows the frequencies of mutations for single-nucleotide (1nt) changes, double-nucleotide (2nt) changes, or changes involving three or more nucleotides (3nt).
    Figure Legend Snippet: Development of HIV-1 resistance to Cas9/sgPBS1. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS1-expressing SupT1 cells were harvested. Viral RNA was extracted and subjected to RT-PCR and sequencing. Each indel in the PBS was registered, and its frequency in the sequenced DNA clones was calculated. The bar graph shows the frequencies of mutations for single-nucleotide (1nt) changes, double-nucleotide (2nt) changes, or changes involving three or more nucleotides (3nt).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay

    Inhibition of HIV-1 infection by Cas9/sgRNA targeting viral PBS. (A) Illustration of the potential mechanisms by which the PBS-targeting Cas9/sgRNA inhibits HIV-1 infection. When the viral RNA carrying the resistant mutation in the PBS is reverse transcribed into viral DNA, the plus strand has the wild-type PBS sequence, thus potentially becoming susceptible to cleavage by Cas9/sgRNA again. vRNA, viral RNA; vDNA, viral DNA. (B) Location of the PBS-targeting sgRNA. The PBS sequence is highlighted in green letters. The PAM is indicated with blue lines and letters. (C) HIV-1 strain NL4-3 was used to infect SupT1 cells that stably express Cas9 and PBS-targeting sgRNA. Expression of Cas9 and viral p24 was examined by immunostaining and flow cytometry. Results of one representative infection experiment are shown. Results of three independent experiments are summarized in the bar graph, with the values of the control set at 100. ***, P
    Figure Legend Snippet: Inhibition of HIV-1 infection by Cas9/sgRNA targeting viral PBS. (A) Illustration of the potential mechanisms by which the PBS-targeting Cas9/sgRNA inhibits HIV-1 infection. When the viral RNA carrying the resistant mutation in the PBS is reverse transcribed into viral DNA, the plus strand has the wild-type PBS sequence, thus potentially becoming susceptible to cleavage by Cas9/sgRNA again. vRNA, viral RNA; vDNA, viral DNA. (B) Location of the PBS-targeting sgRNA. The PBS sequence is highlighted in green letters. The PAM is indicated with blue lines and letters. (C) HIV-1 strain NL4-3 was used to infect SupT1 cells that stably express Cas9 and PBS-targeting sgRNA. Expression of Cas9 and viral p24 was examined by immunostaining and flow cytometry. Results of one representative infection experiment are shown. Results of three independent experiments are summarized in the bar graph, with the values of the control set at 100. ***, P

    Techniques Used: Inhibition, Infection, Mutagenesis, Sequencing, Stable Transfection, Expressing, Immunostaining, Flow Cytometry, Cytometry

    The M3 and M4 PBS mutations resist inhibition by Cas9/sgPBS2. (A) Illustration of the M3 and M4 mutations in the PBS. Also shown are mutations G2557A (S3N in RT) and G4405A (G59E in integrase [IN]), which were detected in viruses that escaped from Cas9/sgPBS2. (B) The M3 and M4 mutants were refractory to Cas9/sgPBS2 in short-term infections. Wild-type and mutated HIV-1 were used to infect control or Cas9/sgPBS2-expressing SupT1 cells. Forty hours later, the levels of infectious HIV-1 in the culture supernatants were determined by infecting the TZM-bl cells. Results shown are the average values from three independent infections. Levels of each virus from the control cells are set at 100. *, P
    Figure Legend Snippet: The M3 and M4 PBS mutations resist inhibition by Cas9/sgPBS2. (A) Illustration of the M3 and M4 mutations in the PBS. Also shown are mutations G2557A (S3N in RT) and G4405A (G59E in integrase [IN]), which were detected in viruses that escaped from Cas9/sgPBS2. (B) The M3 and M4 mutants were refractory to Cas9/sgPBS2 in short-term infections. Wild-type and mutated HIV-1 were used to infect control or Cas9/sgPBS2-expressing SupT1 cells. Forty hours later, the levels of infectious HIV-1 in the culture supernatants were determined by infecting the TZM-bl cells. Results shown are the average values from three independent infections. Levels of each virus from the control cells are set at 100. *, P

    Techniques Used: Inhibition, Expressing

    Development of HIV-1 resistance to Cas9/sgPBS2. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS2-expressing SupT1 cells were collected and sequenced for the PBS DNA. The detected indels are shown, and their frequencies are presented. The bar graphs summarize the percentages of indels involving changes of one nucleotide (1nt), two nucleotides (2nt), or three or more nucleotides (3nt).
    Figure Legend Snippet: Development of HIV-1 resistance to Cas9/sgPBS2. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS2-expressing SupT1 cells were collected and sequenced for the PBS DNA. The detected indels are shown, and their frequencies are presented. The bar graphs summarize the percentages of indels involving changes of one nucleotide (1nt), two nucleotides (2nt), or three or more nucleotides (3nt).

    Techniques Used: Expressing

    The M5 and M6 PBS mutations resist Cas9/sgPBS3 inhibition. (A) Illustration of the M5 and M6 mutations (in red letters) in HIV-1 PBS. (B) Resistance of the M5 and M6 mutants to Cas9/sgPBS3 in short-term infections of SupT1 cells. Results shown are the average values from three independent infection experiments. ***, P
    Figure Legend Snippet: The M5 and M6 PBS mutations resist Cas9/sgPBS3 inhibition. (A) Illustration of the M5 and M6 mutations (in red letters) in HIV-1 PBS. (B) Resistance of the M5 and M6 mutants to Cas9/sgPBS3 in short-term infections of SupT1 cells. Results shown are the average values from three independent infection experiments. ***, P

    Techniques Used: Inhibition, Infection

    40) Product Images from "N6-methyladenine DNA Modification in Glioblastoma"

    Article Title: N6-methyladenine DNA Modification in Glioblastoma

    Journal: Cell

    doi: 10.1016/j.cell.2018.10.006

    Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P
    Figure Legend Snippet: Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P

    Techniques Used: Modification, Dot Blot, Derivative Assay, Mass Spectrometry

    ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P
    Figure Legend Snippet: ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P

    Techniques Used: In Vitro, Recombinant, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Standard Deviation

    41) Product Images from "Joint profiling of DNA methylation and chromatin architecture in single cells"

    Article Title: Joint profiling of DNA methylation and chromatin architecture in single cells

    Journal: Nature methods

    doi: 10.1038/s41592-019-0502-z

    Methyl-HiC simultaneously profiles long-range chromatin interactions and DNA methylome in mouse embryonic stem cells. a. Comparison of contact matrix between in situ Hi-C and Methyl-HiC at different resolutions. Blue squares are loops identified from corresponding dataset at 25kb resolution. Numbers below each map show the maximum contact values. b. Comparison of contact frequency distance decay curve obtained from in situ Hi-C and Methyl-HiC data. c. A snapshot on chromosome 19 showing TADs identified from in situ Hi-C and Methyl-HiC (yellow and green triangles, respectively). d. Pearson correlation for DNA methylation in common CpGs with 15X coverage (n=1,161,200) between WGBS and Methyl-HiC datasets ( p
    Figure Legend Snippet: Methyl-HiC simultaneously profiles long-range chromatin interactions and DNA methylome in mouse embryonic stem cells. a. Comparison of contact matrix between in situ Hi-C and Methyl-HiC at different resolutions. Blue squares are loops identified from corresponding dataset at 25kb resolution. Numbers below each map show the maximum contact values. b. Comparison of contact frequency distance decay curve obtained from in situ Hi-C and Methyl-HiC data. c. A snapshot on chromosome 19 showing TADs identified from in situ Hi-C and Methyl-HiC (yellow and green triangles, respectively). d. Pearson correlation for DNA methylation in common CpGs with 15X coverage (n=1,161,200) between WGBS and Methyl-HiC datasets ( p

    Techniques Used: In Situ, Hi-C, DNA Methylation Assay

    Simultaneous analysis of DNA methylome and chromatin architecture in individual cells by single cell Methyl-HiC a. Workflow of single cell Methyl-HiC. b. Comparison between ensemble contact matrix of single cell Methyl-HiC from 103 serum mESCs and bulk Methyl-HiC on chromosome 2. Contact matrixes are normalized by sequencing coverage and showed at 1Mb resolution. c. Comparison of contact matrixes on chromosome 14 between same number (n=50) of ensemble single cell Hi-C (scHi-C) 15 and single cell Methyl-HiC for mESCs in 2i and serum conditions. Matrix similarity is evaluated by HiCRep 22 at 1Mb resolution. SCC means stratum-adjusted correlation coefficient. d. The comparison of methylation levels distribution between serum (n=103) and 2i (n=47) mESCs from scMethyl-HiC. Boxes are median with quartiles and whiskers extend to 5th and 95th percentile. Red dotted lines show the average methylation levels from previously published data 18 . e. DNA methylation concordance on loop anchors in individual single cell Methyl-HiC datasets. Chromatin loops are identified from bulk Methyl-HiC using HiCCUPS (n=3110). Boxes are median with quartiles and whiskers extend to 5th and 95th percentile.
    Figure Legend Snippet: Simultaneous analysis of DNA methylome and chromatin architecture in individual cells by single cell Methyl-HiC a. Workflow of single cell Methyl-HiC. b. Comparison between ensemble contact matrix of single cell Methyl-HiC from 103 serum mESCs and bulk Methyl-HiC on chromosome 2. Contact matrixes are normalized by sequencing coverage and showed at 1Mb resolution. c. Comparison of contact matrixes on chromosome 14 between same number (n=50) of ensemble single cell Hi-C (scHi-C) 15 and single cell Methyl-HiC for mESCs in 2i and serum conditions. Matrix similarity is evaluated by HiCRep 22 at 1Mb resolution. SCC means stratum-adjusted correlation coefficient. d. The comparison of methylation levels distribution between serum (n=103) and 2i (n=47) mESCs from scMethyl-HiC. Boxes are median with quartiles and whiskers extend to 5th and 95th percentile. Red dotted lines show the average methylation levels from previously published data 18 . e. DNA methylation concordance on loop anchors in individual single cell Methyl-HiC datasets. Chromatin loops are identified from bulk Methyl-HiC using HiCCUPS (n=3110). Boxes are median with quartiles and whiskers extend to 5th and 95th percentile.

    Techniques Used: Sequencing, Hi-C, Methylation, DNA Methylation Assay

    Single cell Methyl-HiC reveals heterogeneity of cultured mouse embryonic stem cells. a. t-SNE visualization of unsupervised clustering results according to DNA methylation from individual single cell Methyl-HiC dataset (n=150). Methylation level is calculated in non-overlapping 1Mb bins. b. Unsupervised clustering of aggregated DNA methylome in subgroups of serum mESCs with tissue-specific methylomes in mouse embryonic development. c. Pearson’s correlation matrixes from different cell clusters. Similar numbers of cells are randomly selected to plot the matrixes (n=24, 26, and 24). The left map shows the bulk in situ Hi-C matrixes from serum and 2i mESCs, respectively. Pearson’s correlation is calculated under 1Mb resolution. Color ranges have been set to the same scale. d. GO biological process terms predicted by GREAT for DMRs (n=99) switching from Compartment B in Cluster 2 to Compartment A in Cluster 3. e. GO biological process terms of genes (n=672) switching from Compartment B in Cluster 2 to Compartment A in Cluster 3. P-values are from modified Fisher Exact test for gene-enrichment analysis. f. Snapshot of HoxD cluster genes in differential compartments between Cluster 2 and 3. Hi-C matrix are from bulk in situ Hi-C of the same cell line to show the chromosome organization in nearby region. Two tracks below the HiC matrix are eigenvector decomposition. The positive/negative values correspond to compartment A/B, respectively. DMRs between Cluster 2 and Cluster 3 are also plotted.
    Figure Legend Snippet: Single cell Methyl-HiC reveals heterogeneity of cultured mouse embryonic stem cells. a. t-SNE visualization of unsupervised clustering results according to DNA methylation from individual single cell Methyl-HiC dataset (n=150). Methylation level is calculated in non-overlapping 1Mb bins. b. Unsupervised clustering of aggregated DNA methylome in subgroups of serum mESCs with tissue-specific methylomes in mouse embryonic development. c. Pearson’s correlation matrixes from different cell clusters. Similar numbers of cells are randomly selected to plot the matrixes (n=24, 26, and 24). The left map shows the bulk in situ Hi-C matrixes from serum and 2i mESCs, respectively. Pearson’s correlation is calculated under 1Mb resolution. Color ranges have been set to the same scale. d. GO biological process terms predicted by GREAT for DMRs (n=99) switching from Compartment B in Cluster 2 to Compartment A in Cluster 3. e. GO biological process terms of genes (n=672) switching from Compartment B in Cluster 2 to Compartment A in Cluster 3. P-values are from modified Fisher Exact test for gene-enrichment analysis. f. Snapshot of HoxD cluster genes in differential compartments between Cluster 2 and 3. Hi-C matrix are from bulk in situ Hi-C of the same cell line to show the chromosome organization in nearby region. Two tracks below the HiC matrix are eigenvector decomposition. The positive/negative values correspond to compartment A/B, respectively. DMRs between Cluster 2 and Cluster 3 are also plotted.

    Techniques Used: Cell Culture, DNA Methylation Assay, Methylation, In Situ, Hi-C, Modification

    42) Product Images from "16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles"

    Article Title: 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

    Journal: Microbiome

    doi: 10.1186/2049-2618-2-31

    Normalization of PCR amplification by choosing PCR cycle number close to the Ct value. Throat swabs from healthy volunteers were extracted with Qiagen DNeasy (D) or MO BIO PowerSoil (M). DNA extracts were subjected to 16S gene TaqMan qPCR and subsequent PCR using a cycle number of 20, 25, or 30 based on individual sample's qPCR Ct value. PCR amplicons were quantified by PicoGreen dsDNA assay. DNA concentrations for DNA extracts ( blue markers and axis ) and PCR amplicons ( red markers and axis ) are shown in 16S gene copies/μl in the same scale. DNA sample name, 08S1M as an example, depicts the subject number (08), swab number (S1), and extraction method (M).
    Figure Legend Snippet: Normalization of PCR amplification by choosing PCR cycle number close to the Ct value. Throat swabs from healthy volunteers were extracted with Qiagen DNeasy (D) or MO BIO PowerSoil (M). DNA extracts were subjected to 16S gene TaqMan qPCR and subsequent PCR using a cycle number of 20, 25, or 30 based on individual sample's qPCR Ct value. PCR amplicons were quantified by PicoGreen dsDNA assay. DNA concentrations for DNA extracts ( blue markers and axis ) and PCR amplicons ( red markers and axis ) are shown in 16S gene copies/μl in the same scale. DNA sample name, 08S1M as an example, depicts the subject number (08), swab number (S1), and extraction method (M).

    Techniques Used: Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Picogreen Assay

    Storage temperature comparisons for Qiagen DNeasy and MO BIO PowerSoil DNA extraction methods for the mock bacterial community HM-280. Identical samples from the mock community were stored at four different temperatures and extracted after 4 weeks using the two methods, Qiagen DNeasy and MO BIO PowerSoil. Microbial profiles at the genus level were estimated using QIIME. Overall, samples were well preserved at lower temperatures for both methods, whereas significant differences were observed at 37°C.
    Figure Legend Snippet: Storage temperature comparisons for Qiagen DNeasy and MO BIO PowerSoil DNA extraction methods for the mock bacterial community HM-280. Identical samples from the mock community were stored at four different temperatures and extracted after 4 weeks using the two methods, Qiagen DNeasy and MO BIO PowerSoil. Microbial profiles at the genus level were estimated using QIIME. Overall, samples were well preserved at lower temperatures for both methods, whereas significant differences were observed at 37°C.

    Techniques Used: DNA Extraction

    43) Product Images from "Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates"

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.5

    Comparison of vector transduction titers measured following in situ versus spin column (DNeasy) DNA extraction. 293T-MLV-DCSIGN assay cells were transduced with equivalent amounts of integration-competent or -deficient vector particles (as measured by vector genomes). Following an overnight incubation, DNA was isolated, and samples were analyzed by qPCR to detect vector sequence. ( a ) Extraction performed using in situ method. Each bar represents the mean ± SD of nine transductions performed in parallel ( n = 3 qPCR replicates/transduction). ( b ) Extraction performed using DNeasy column (5 × 10 4 cells loaded per column). Each bar represents the mean ± SD of qPCR triplicates for a single transduction; two transductions were performed in parallel for each vector tested (replicates 1, 2). Particle-to-infectivity (P:I) ratios were calculated by dividing the physical particle titer (measured by RNA genome content per vector) by the measured transduction titer. Results in both panels are representative of two independent experiments. TU, transduction units.
    Figure Legend Snippet: Comparison of vector transduction titers measured following in situ versus spin column (DNeasy) DNA extraction. 293T-MLV-DCSIGN assay cells were transduced with equivalent amounts of integration-competent or -deficient vector particles (as measured by vector genomes). Following an overnight incubation, DNA was isolated, and samples were analyzed by qPCR to detect vector sequence. ( a ) Extraction performed using in situ method. Each bar represents the mean ± SD of nine transductions performed in parallel ( n = 3 qPCR replicates/transduction). ( b ) Extraction performed using DNeasy column (5 × 10 4 cells loaded per column). Each bar represents the mean ± SD of qPCR triplicates for a single transduction; two transductions were performed in parallel for each vector tested (replicates 1, 2). Particle-to-infectivity (P:I) ratios were calculated by dividing the physical particle titer (measured by RNA genome content per vector) by the measured transduction titer. Results in both panels are representative of two independent experiments. TU, transduction units.

    Techniques Used: Plasmid Preparation, Transduction, In Situ, DNA Extraction, Incubation, Isolation, Real-time Polymerase Chain Reaction, Sequencing, Infection

    44) Product Images from "Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)"

    Article Title: Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2014.52.3.305

    Detection limit of Azumiobodo hoyamushi 18S rDNA LAMP assays (A). LAMP assays were performed using serial dilutions of A. hoyamushi genomic DNA (1 ng to 1 fg per reaction). Distilled water was used as a negative control. LAMP products were visualized by gel electrophoresis (B) and using Loopamp® fluorescent detection reagent (FD) (C). (B, C) Lane M, 100-bp DNA marker; lane 1, 1 ng; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg of A. hoyamushi genomic DNA; lane 8, distilled water; and lane 9, LAMP product after Mbo I digestion. (D-E) A. hoyamushi at a density of 1×10 3 parasites/µl was serially diluted and tested (D) using the LAMP assay (D) and by PCR (E) using F3 and B3 primers. Lane M, 100-bp DNA marker; lane 1, 1,000; lane 2, 100; lane 3, 10; lane 4, 1; lane 5, 0.1; lane 6, 0.01 of parasites per reaction; lane 7, distilled water. A. hoyamushi genomic DNA was prepared using DNeasy tissue kits (Qiagen) from in vitro cultured A. hoyamushi species [ 9 ] which were kindly provided by Dr. Kyung Il Park (Kunsan National University, Gunsan, Korea).
    Figure Legend Snippet: Detection limit of Azumiobodo hoyamushi 18S rDNA LAMP assays (A). LAMP assays were performed using serial dilutions of A. hoyamushi genomic DNA (1 ng to 1 fg per reaction). Distilled water was used as a negative control. LAMP products were visualized by gel electrophoresis (B) and using Loopamp® fluorescent detection reagent (FD) (C). (B, C) Lane M, 100-bp DNA marker; lane 1, 1 ng; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg of A. hoyamushi genomic DNA; lane 8, distilled water; and lane 9, LAMP product after Mbo I digestion. (D-E) A. hoyamushi at a density of 1×10 3 parasites/µl was serially diluted and tested (D) using the LAMP assay (D) and by PCR (E) using F3 and B3 primers. Lane M, 100-bp DNA marker; lane 1, 1,000; lane 2, 100; lane 3, 10; lane 4, 1; lane 5, 0.1; lane 6, 0.01 of parasites per reaction; lane 7, distilled water. A. hoyamushi genomic DNA was prepared using DNeasy tissue kits (Qiagen) from in vitro cultured A. hoyamushi species [ 9 ] which were kindly provided by Dr. Kyung Il Park (Kunsan National University, Gunsan, Korea).

    Techniques Used: Negative Control, Nucleic Acid Electrophoresis, Marker, Lamp Assay, Polymerase Chain Reaction, In Vitro, Cell Culture

    45) Product Images from "Isolation and preservation of schistosome eggs and larvae in RNAlater® facilitates genetic profiling of individuals"

    Article Title: Isolation and preservation of schistosome eggs and larvae in RNAlater® facilitates genetic profiling of individuals

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-2-50

    Schematic of the RNA later ® preservation and gDNA extraction of individual schistosome larval stages and eggs . ATL and AL are lysis buffers supplied in the Qiagen DNeasy Blood and Tissue Kit. *These were the longest times and highest temperatures tested but it is expected that the samples can be preserved for much longer.
    Figure Legend Snippet: Schematic of the RNA later ® preservation and gDNA extraction of individual schistosome larval stages and eggs . ATL and AL are lysis buffers supplied in the Qiagen DNeasy Blood and Tissue Kit. *These were the longest times and highest temperatures tested but it is expected that the samples can be preserved for much longer.

    Techniques Used: Preserving, Lysis

    46) Product Images from "Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma"

    Article Title: Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011367

    Luciferase activity and PCR analysis of tumors from mice in the pCSI/pLiLucB group provide no evidence of ϕC31 integrase activity. ( a ) Protein extracts were prepared and the luciferase activity was measured in absolute counts per second (CPS). Controls included HeLa cells given FuGene 6 alone [HeLa (-)] or the CMV-luciferase plasmid pNBL2 via FuGene 6 [HeLa (+)], the normal-appearing part of the tumor-ridden liver taken from either a saline-injected mouse [Liver (-)] and pCSI/pLiLucB-injected mouse [Liver (+)]. Eight tumor samples (Tumor 1 through 8) and one metastasis (Met 1) that were obtained from four animals were also analyzed. The error bars give standard error of the mean for four replicates of each sample. ( b ) PCR analysis to detect the pLiLucB plasmid by amplification of the luciferase transgene. Plasmid DNA (20 ng pLiLucB) and no DNA controls show specific amplification of luciferase only in the reaction containing plasmid. One mouse each from the saline-only and pCSmI/pLiLucB groups was analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues (none found). Three mice in the pCSI/pLiLucB group were analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues. Luciferase could be detected in 2/3 normal-appearing liver samples and none of the tumors. ( c ) PCR analysis for integration at the mpsL1 pseudo attP site was done on 18 tumors (lanes 5 through 22) and one metastasis (lane 23) taken from nine mice given pCSI/pLiLucB by hydrodynamic injection. Controls included no DNA (1 st round, lane 1 and 2 nd round, lane 25), and a DNeasy performed on no tissue (lane 2) to show no contamination from the DNA isolation procedure. Normal-appearing liver from a mouse in the pCSI/pLiLucB group (lane 3) served as the positive control. DNA isolated from a tumor in the saline-only group served as the negative control (lane 4). PCR for the GAPDH gene showed that sufficient DNA was added to all reactions. Seven tumors and one metastasis were subjected to the analysis in both a and c . Six tumors were analyzed by all assays ( a , b and c ).
    Figure Legend Snippet: Luciferase activity and PCR analysis of tumors from mice in the pCSI/pLiLucB group provide no evidence of ϕC31 integrase activity. ( a ) Protein extracts were prepared and the luciferase activity was measured in absolute counts per second (CPS). Controls included HeLa cells given FuGene 6 alone [HeLa (-)] or the CMV-luciferase plasmid pNBL2 via FuGene 6 [HeLa (+)], the normal-appearing part of the tumor-ridden liver taken from either a saline-injected mouse [Liver (-)] and pCSI/pLiLucB-injected mouse [Liver (+)]. Eight tumor samples (Tumor 1 through 8) and one metastasis (Met 1) that were obtained from four animals were also analyzed. The error bars give standard error of the mean for four replicates of each sample. ( b ) PCR analysis to detect the pLiLucB plasmid by amplification of the luciferase transgene. Plasmid DNA (20 ng pLiLucB) and no DNA controls show specific amplification of luciferase only in the reaction containing plasmid. One mouse each from the saline-only and pCSmI/pLiLucB groups was analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues (none found). Three mice in the pCSI/pLiLucB group were analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues. Luciferase could be detected in 2/3 normal-appearing liver samples and none of the tumors. ( c ) PCR analysis for integration at the mpsL1 pseudo attP site was done on 18 tumors (lanes 5 through 22) and one metastasis (lane 23) taken from nine mice given pCSI/pLiLucB by hydrodynamic injection. Controls included no DNA (1 st round, lane 1 and 2 nd round, lane 25), and a DNeasy performed on no tissue (lane 2) to show no contamination from the DNA isolation procedure. Normal-appearing liver from a mouse in the pCSI/pLiLucB group (lane 3) served as the positive control. DNA isolated from a tumor in the saline-only group served as the negative control (lane 4). PCR for the GAPDH gene showed that sufficient DNA was added to all reactions. Seven tumors and one metastasis were subjected to the analysis in both a and c . Six tumors were analyzed by all assays ( a , b and c ).

    Techniques Used: Luciferase, Activity Assay, Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, Injection, Amplification, DNA Extraction, Positive Control, Isolation, Negative Control

    47) Product Images from "Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿"

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03050-09

    Agarose gel electrophoresis of purified enterococcal DNA. (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).
    Figure Legend Snippet: Agarose gel electrophoresis of purified enterococcal DNA. (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).

    Techniques Used: Agarose Gel Electrophoresis, Purification, Plasmid Preparation

    48) Product Images from "Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization, et al. Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization"

    Article Title: Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization, et al. Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

    Journal: Ecology and Evolution

    doi: 10.1002/ece3.4843

    DNA yield from seven extraction protocols (DNeasy: Qiagen DNeasy Blood Tissue Kit; MO BIO PW: MO BIO PowerWater DNA Isolation Kit; MO BIO PMS: MO BIO PowerMax Soil DNA Isolation Kit; Dnature: Presto™ Mini gDNA Bacteria Kit; PCI: phenol‐chloroform‐isoamyl extraction procedure; Silica: Silica extraction procedure; and Magnetic beads: Magnetic beads extraction procedure) obtained from 1,000 ml technical replicates filtered through 1.2‐µm cellulose‐nitrate filters. Error bars show 95% confidence intervals. Letters indicate significant differences among groups based on Tukey–Kramer's test (α = 0.05)
    Figure Legend Snippet: DNA yield from seven extraction protocols (DNeasy: Qiagen DNeasy Blood Tissue Kit; MO BIO PW: MO BIO PowerWater DNA Isolation Kit; MO BIO PMS: MO BIO PowerMax Soil DNA Isolation Kit; Dnature: Presto™ Mini gDNA Bacteria Kit; PCI: phenol‐chloroform‐isoamyl extraction procedure; Silica: Silica extraction procedure; and Magnetic beads: Magnetic beads extraction procedure) obtained from 1,000 ml technical replicates filtered through 1.2‐µm cellulose‐nitrate filters. Error bars show 95% confidence intervals. Letters indicate significant differences among groups based on Tukey–Kramer's test (α = 0.05)

    Techniques Used: DNA Extraction, Magnetic Beads

    The average (a) OTU and (b) taxon richness obtained per replicate for each of the four assays between the optimal (blue; 1.2‐µm cellulose‐nitrate filter and Qiagen's DNeasy Blood Tissue Kit) and low‐performance (gold; 1.2‐µm polycarbonate filter and MO BIO's PowerMax Soil) protocol. Error bars show 95% confidence intervals. T test significance is depicted by: *** p
    Figure Legend Snippet: The average (a) OTU and (b) taxon richness obtained per replicate for each of the four assays between the optimal (blue; 1.2‐µm cellulose‐nitrate filter and Qiagen's DNeasy Blood Tissue Kit) and low‐performance (gold; 1.2‐µm polycarbonate filter and MO BIO's PowerMax Soil) protocol. Error bars show 95% confidence intervals. T test significance is depicted by: *** p

    Techniques Used:

    Observed taxa in each of the four assays. Filled and unfilled rectangles indicate taxon presence or absence. Taxa show a pair of rectangles per assay, representing the low‐performance (PCPMS; 1.2‐µm polycarbonate filter and MO BIO's PowerMax Soil) and the optimal (CNQ; 1.2‐µm cellulose‐nitrate filter and Qiagen's DNeasy Blood Tissue Kit) treatment. Rectangular presence indicates the amplification range of each assay
    Figure Legend Snippet: Observed taxa in each of the four assays. Filled and unfilled rectangles indicate taxon presence or absence. Taxa show a pair of rectangles per assay, representing the low‐performance (PCPMS; 1.2‐µm polycarbonate filter and MO BIO's PowerMax Soil) and the optimal (CNQ; 1.2‐µm cellulose‐nitrate filter and Qiagen's DNeasy Blood Tissue Kit) treatment. Rectangular presence indicates the amplification range of each assay

    Techniques Used: Amplification

    49) Product Images from "Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species"

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

    Journal:

    doi:

    Comparison of DNA extraction methods for detection of Aphelenchoides fragariae in naturally infected Asplenium nidus (Bird's nest fern) plant tissue using species-specific PCR primers. Lane 1: healthy A. nidus extracted with Qiagen Dneasy Plant Mini Kit;
    Figure Legend Snippet: Comparison of DNA extraction methods for detection of Aphelenchoides fragariae in naturally infected Asplenium nidus (Bird's nest fern) plant tissue using species-specific PCR primers. Lane 1: healthy A. nidus extracted with Qiagen Dneasy Plant Mini Kit;

    Techniques Used: DNA Extraction, Infection, Polymerase Chain Reaction

    50) Product Images from "Methods to maximise recovery of environmental DNA from water samples"

    Article Title: Methods to maximise recovery of environmental DNA from water samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179251

    Experimental design for Experiment 2A to investigate which DNA extraction kit-filter paper combination would give the best DNA yield. N refers to number of technical replicates. CN, MCE, PES, PCTE and GF refer to the types of filter papers whereas DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.
    Figure Legend Snippet: Experimental design for Experiment 2A to investigate which DNA extraction kit-filter paper combination would give the best DNA yield. N refers to number of technical replicates. CN, MCE, PES, PCTE and GF refer to the types of filter papers whereas DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.

    Techniques Used: DNA Extraction

    DNA yield from eight DNA extraction kit-filter paper combinations from samples in aquaria with UV-sterilized water. Clear bars used DNeasy extraction kit while shaded grey bars used the PowerWater kit. CN = Cellulose Nitrate, MCE = Mixed Cellulose Ester, PES = Polyethersulfone, PCTE = Polycarbonate track-etched. Error bars show the ±2 standard deviation of the mean.
    Figure Legend Snippet: DNA yield from eight DNA extraction kit-filter paper combinations from samples in aquaria with UV-sterilized water. Clear bars used DNeasy extraction kit while shaded grey bars used the PowerWater kit. CN = Cellulose Nitrate, MCE = Mixed Cellulose Ester, PES = Polyethersulfone, PCTE = Polycarbonate track-etched. Error bars show the ±2 standard deviation of the mean.

    Techniques Used: DNA Extraction, Standard Deviation

    Experimental design for Experiment 2B investigating DNA yields in five different DNA extraction kit-filter paper combination from stream water. N refers to number of technical replicates. CN, MCE, PES, PCTE and GF refer to the types of filter papers whereas DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.
    Figure Legend Snippet: Experimental design for Experiment 2B investigating DNA yields in five different DNA extraction kit-filter paper combination from stream water. N refers to number of technical replicates. CN, MCE, PES, PCTE and GF refer to the types of filter papers whereas DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.

    Techniques Used: DNA Extraction

    Experimental design used to compare five different combinations of capture, preservation and DNA extraction methods. N is the number of technical replicates and each replicate represents one 250 ml water sample. DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.
    Figure Legend Snippet: Experimental design used to compare five different combinations of capture, preservation and DNA extraction methods. N is the number of technical replicates and each replicate represents one 250 ml water sample. DNeasy and PowerWater (PW) refer to the two DNA extraction kits used in the experiment.

    Techniques Used: Preserving, DNA Extraction

    DNA yield from five different extraction kit-filter paper combinations from stream water samples. Clear bars used DNeasy extraction kit while shaded grey bars used the PowerWater kit. Error bars show the ±2 standard deviation of the mean.
    Figure Legend Snippet: DNA yield from five different extraction kit-filter paper combinations from stream water samples. Clear bars used DNeasy extraction kit while shaded grey bars used the PowerWater kit. Error bars show the ±2 standard deviation of the mean.

    Techniques Used: Standard Deviation

    51) Product Images from "Loop-mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena)"

    Article Title: Loop-mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184349

    Application of the LAMP assay on a stranded harbour porpoise. Gel electrophoresis of all LAMP products of the stranded harbour porpoise. Directly tested MSwab ™ samples (without DNA isolation) and DNA isolation of the MSwab ™ medium using DNeasy ® blood and tissue kit.
    Figure Legend Snippet: Application of the LAMP assay on a stranded harbour porpoise. Gel electrophoresis of all LAMP products of the stranded harbour porpoise. Directly tested MSwab ™ samples (without DNA isolation) and DNA isolation of the MSwab ™ medium using DNeasy ® blood and tissue kit.

    Techniques Used: Lamp Assay, Nucleic Acid Electrophoresis, DNA Extraction

    52) Product Images from "Evaluation of Real-time PCR Based on SYBR Green I Fluorescent Dye for Detection of Bacillus Anthracis Strains in Biological Samples"

    Article Title: Evaluation of Real-time PCR Based on SYBR Green I Fluorescent Dye for Detection of Bacillus Anthracis Strains in Biological Samples

    Journal: Journal of Veterinary Research

    doi: 10.2478/jvetres-2018-0075

    Determination of the sensitivity of SYBR Green I real-time PCR with rpoB-F/R primers for the detection of B. anthracis strain 1/47 in liver (a) and blood (b) samples using a DNeasy Blood and Tissue Kit for DNA isolation
    Figure Legend Snippet: Determination of the sensitivity of SYBR Green I real-time PCR with rpoB-F/R primers for the detection of B. anthracis strain 1/47 in liver (a) and blood (b) samples using a DNeasy Blood and Tissue Kit for DNA isolation

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, DNA Extraction

    Determination of the sensitivity of SYBR Green I real-time PCR with capC-F/R primers for the detection of B. anthracis strain 1/47 in liver (a) and blood (b) samples using a DNeasy Blood and Tissue Kit for DNA isolation
    Figure Legend Snippet: Determination of the sensitivity of SYBR Green I real-time PCR with capC-F/R primers for the detection of B. anthracis strain 1/47 in liver (a) and blood (b) samples using a DNeasy Blood and Tissue Kit for DNA isolation

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, DNA Extraction

    53) Product Images from "Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04® via Chip-Based Digital PCR"

    Article Title: Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04® via Chip-Based Digital PCR

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00704

    Qiagen DNeasy replicate isolations of DNA from NCFM overnight cells not treated with PMA. Columns with differing letter are significantly different from each other.
    Figure Legend Snippet: Qiagen DNeasy replicate isolations of DNA from NCFM overnight cells not treated with PMA. Columns with differing letter are significantly different from each other.

    Techniques Used:

    54) Product Images from "Mitochondrial group I and group II introns in the sponge orders Agelasida and Axinellida"

    Article Title: Mitochondrial group I and group II introns in the sponge orders Agelasida and Axinellida

    Journal: BMC Evolutionary Biology

    doi: 10.1186/s12862-015-0556-1

    Predicted secondary structure of A. oroides , A. polypoides and C. verrucosa introns 723 (Group I). The exon and intron bases are indicated in lower-case and upper-case letters, respectively. The conserved sequences (P, Q, R, S) of the intron core and the base-paired regions P1–P10 follow the standard Group I intron scheme [ 47 ]. Regions with substantial structural differences among species are indicated with a colored background
    Figure Legend Snippet: Predicted secondary structure of A. oroides , A. polypoides and C. verrucosa introns 723 (Group I). The exon and intron bases are indicated in lower-case and upper-case letters, respectively. The conserved sequences (P, Q, R, S) of the intron core and the base-paired regions P1–P10 follow the standard Group I intron scheme [ 47 ]. Regions with substantial structural differences among species are indicated with a colored background

    Techniques Used:

    Predicted secondary structure of A. oroides and A. polypoides introns 870 (Group I). The exon and intron bases are indicated in lower-case and upper-case letters, respectively. The conserved sequences (P, Q, R, S) of the intron core and the base-paired regions P1–P10 follow the standard Group I intron scheme [ 47 ]. Regions with substantial structural differences among species are indicated with a colored background
    Figure Legend Snippet: Predicted secondary structure of A. oroides and A. polypoides introns 870 (Group I). The exon and intron bases are indicated in lower-case and upper-case letters, respectively. The conserved sequences (P, Q, R, S) of the intron core and the base-paired regions P1–P10 follow the standard Group I intron scheme [ 47 ]. Regions with substantial structural differences among species are indicated with a colored background

    Techniques Used:

    55) Product Images from "Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine"

    Article Title: Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154949

    Fluorescence activated nuclear sorting (FANS) of three classes of visceral adipose tissue cellular nuclei immuno-stained for PPARg2. A. Histogram of PPARg2-Neg, -Low, -Med. and -High stained nuclei from sorting experiment (log scale) gated for DNA content (DAPI subset, S1B Fig ) and forward and side light scattering ( S1A Fig ). B . PPARg2 transcript levels were assayed among the four fractions of nuclei by qRT-PCR. C, D, E, F. Merged immunofluorescence microscope images of four isolated fractions of nuclei without re-staining. G. Comparison of the average nuclear area for the four fractions from one experiment (N = 100). There were statistically significant differences between all of the fractions of nuclear areas except for between PPARg2-Low and PPARg2-Neg fractions. H. PPARg2-High nuclei from image H showing the DAPI staining alone to reveal decondensed nuclei, however, some strongly stained PPARg2-High nuclei are small reflecting some heterogeneity in their morphology, as indicated by white arrows. For antibodies see S1 Table . A p value of P
    Figure Legend Snippet: Fluorescence activated nuclear sorting (FANS) of three classes of visceral adipose tissue cellular nuclei immuno-stained for PPARg2. A. Histogram of PPARg2-Neg, -Low, -Med. and -High stained nuclei from sorting experiment (log scale) gated for DNA content (DAPI subset, S1B Fig ) and forward and side light scattering ( S1A Fig ). B . PPARg2 transcript levels were assayed among the four fractions of nuclei by qRT-PCR. C, D, E, F. Merged immunofluorescence microscope images of four isolated fractions of nuclei without re-staining. G. Comparison of the average nuclear area for the four fractions from one experiment (N = 100). There were statistically significant differences between all of the fractions of nuclear areas except for between PPARg2-Low and PPARg2-Neg fractions. H. PPARg2-High nuclei from image H showing the DAPI staining alone to reveal decondensed nuclei, however, some strongly stained PPARg2-High nuclei are small reflecting some heterogeneity in their morphology, as indicated by white arrows. For antibodies see S1 Table . A p value of P

    Techniques Used: Fluorescence, Staining, Quantitative RT-PCR, Immunofluorescence, Microscopy, Isolation

    Distribution of 5hmC among adipose tissue nuclei. IFM and FNC were used to examine 5hmC levels among visceral adipose tissue nuclei. A. A field of VAT nuclei examined with various combinations of DAPI staining for DNA, and immunostaining with mouse anti-PPARg2 + goat anti-mouse Alexafluor488 and rabbit anti-5hmC + goat anti-rabbit Alexafluor633. White arrows indicate those large, decondensed nuclei that are stained strongly for both 5hmC and PPARg2. B. Flow Cytometry of VAT nuclei immunostained as in A. C. Goat anti-rabbit secondary antibody used in B shows only modest background staining of nuclei. Nuclei were gated for DAPI ( > 2C DNA content) and size and shape by light scattering as in S1 Fig . For antibodies see S1 Table .
    Figure Legend Snippet: Distribution of 5hmC among adipose tissue nuclei. IFM and FNC were used to examine 5hmC levels among visceral adipose tissue nuclei. A. A field of VAT nuclei examined with various combinations of DAPI staining for DNA, and immunostaining with mouse anti-PPARg2 + goat anti-mouse Alexafluor488 and rabbit anti-5hmC + goat anti-rabbit Alexafluor633. White arrows indicate those large, decondensed nuclei that are stained strongly for both 5hmC and PPARg2. B. Flow Cytometry of VAT nuclei immunostained as in A. C. Goat anti-rabbit secondary antibody used in B shows only modest background staining of nuclei. Nuclei were gated for DAPI ( > 2C DNA content) and size and shape by light scattering as in S1 Fig . For antibodies see S1 Table .

    Techniques Used: Staining, Immunostaining, Flow Cytometry, Cytometry

    56) Product Images from "Identification and validation of a Schistosoma japonicum U6 promoter"

    Article Title: Identification and validation of a Schistosoma japonicum U6 promoter

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2207-4

    sjU6 promoter is active in S. japonicum . Schistosomula were transduced 14 dpi with pHBLV-sgRNA970 lentivirus for 48 h and cultured for additional 48 h after viral exposure. Lane M: DL2000 DNA marker; Lane 1: amplification of the 445 bp sjU6-sgRNA970 expression cassette from gDNA of untrasduced schistosomula; Lane 2: amplification of the 445 bp sjU6-sgRNA970 expression cassette from transduced schistosomula gDNA; Lane3: amplification of the 98 bp sgRNA970 from untransduced schistosomula cDNA; Lane4: amplification of the 98 bp sgRNA970 from transduced schistosomula RNA treated with DNAseI, but without reverse transcription; Lane 5: amplification of the PSMD internal control gene from cDNA synthesized from transduced schistosomula RNA treated with DNAseI; Lane 6, amplification of the 98 bp sgRNA970 from cDNA synthesized from transduced schistosomula RNA treated with DNAseI; Lane 7: amplification of the 445 bp sjU6-sgRNA970 expression cassette from cDNA synthesized from transduced schistosomula RNA treated with DNAseI
    Figure Legend Snippet: sjU6 promoter is active in S. japonicum . Schistosomula were transduced 14 dpi with pHBLV-sgRNA970 lentivirus for 48 h and cultured for additional 48 h after viral exposure. Lane M: DL2000 DNA marker; Lane 1: amplification of the 445 bp sjU6-sgRNA970 expression cassette from gDNA of untrasduced schistosomula; Lane 2: amplification of the 445 bp sjU6-sgRNA970 expression cassette from transduced schistosomula gDNA; Lane3: amplification of the 98 bp sgRNA970 from untransduced schistosomula cDNA; Lane4: amplification of the 98 bp sgRNA970 from transduced schistosomula RNA treated with DNAseI, but without reverse transcription; Lane 5: amplification of the PSMD internal control gene from cDNA synthesized from transduced schistosomula RNA treated with DNAseI; Lane 6, amplification of the 98 bp sgRNA970 from cDNA synthesized from transduced schistosomula RNA treated with DNAseI; Lane 7: amplification of the 445 bp sjU6-sgRNA970 expression cassette from cDNA synthesized from transduced schistosomula RNA treated with DNAseI

    Techniques Used: Cell Culture, Marker, Amplification, Expressing, Synthesized

    57) Product Images from "Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines"

    Article Title: Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines

    Journal: Journal of Cancer Research and Clinical Oncology

    doi: 10.1007/s00432-016-2308-z

    Effect of exogenous ligand application on trastuzumab sensitivity in gastric cancer cell lines. GSU, H111TC, KATOIII and MKN45 cells were treated for 3 days with trastuzumab alone (0, 1, 20 µg/ml) and/or different HER receptor ligands (AREG: 15 ng/ml; EGF: 0.1 ng/ml; HB-EGF: 0.4 ng/ml). The metabolic activity of the cells was measured using the WST-1 cell proliferation assay. In GSU cells, HB-EGF but not AREG and EGF was effective in rescuing the cells from trastuzumab treatment. In H111TC, a similar but not significant trend was observed. No effect of either ligand was detected for the trastuzumab-resistant cell lines KATOIII and MKN45. The mean value of three independent experiments is shown. For better readability only p values referring to the control are shown. For all significant p values, please refer to Table 2 . p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively
    Figure Legend Snippet: Effect of exogenous ligand application on trastuzumab sensitivity in gastric cancer cell lines. GSU, H111TC, KATOIII and MKN45 cells were treated for 3 days with trastuzumab alone (0, 1, 20 µg/ml) and/or different HER receptor ligands (AREG: 15 ng/ml; EGF: 0.1 ng/ml; HB-EGF: 0.4 ng/ml). The metabolic activity of the cells was measured using the WST-1 cell proliferation assay. In GSU cells, HB-EGF but not AREG and EGF was effective in rescuing the cells from trastuzumab treatment. In H111TC, a similar but not significant trend was observed. No effect of either ligand was detected for the trastuzumab-resistant cell lines KATOIII and MKN45. The mean value of three independent experiments is shown. For better readability only p values referring to the control are shown. For all significant p values, please refer to Table 2 . p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively

    Techniques Used: Activity Assay, Proliferation Assay

    Effect of trastuzumab treatment on the cell proliferation of gastric cancer cell lines. The gastric cell lines AGS, GSU, H111TC, HGC-27, Hs746T, KATOIII, LMSU, MKN1, MKN7, MKN28 and MKN45 were treated for 72 h with the indicated amounts of trastuzumab (0/0.1/1/10/20/40 µg/ml), a solvent control (Sol), an isotype control (ISO) or isotype solvent control (ISO-Sol). Afterwards, the metabolic activity of the cell lines was determined via WST-1 cell proliferation assay. Only GSU and H111TC cells were trastuzumab sensitive ( dark grey diagrams ). The mean value of at least three independent experiments is shown. p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively
    Figure Legend Snippet: Effect of trastuzumab treatment on the cell proliferation of gastric cancer cell lines. The gastric cell lines AGS, GSU, H111TC, HGC-27, Hs746T, KATOIII, LMSU, MKN1, MKN7, MKN28 and MKN45 were treated for 72 h with the indicated amounts of trastuzumab (0/0.1/1/10/20/40 µg/ml), a solvent control (Sol), an isotype control (ISO) or isotype solvent control (ISO-Sol). Afterwards, the metabolic activity of the cell lines was determined via WST-1 cell proliferation assay. Only GSU and H111TC cells were trastuzumab sensitive ( dark grey diagrams ). The mean value of at least three independent experiments is shown. p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively

    Techniques Used: Activity Assay, Proliferation Assay

    58) Product Images from "Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines"

    Article Title: Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines

    Journal: Journal of Cancer Research and Clinical Oncology

    doi: 10.1007/s00432-016-2308-z

    Effect of exogenous ligand application on trastuzumab sensitivity in gastric cancer cell lines. GSU, H111TC, KATOIII and MKN45 cells were treated for 3 days with trastuzumab alone (0, 1, 20 µg/ml) and/or different HER receptor ligands (AREG: 15 ng/ml; EGF: 0.1 ng/ml; HB-EGF: 0.4 ng/ml). The metabolic activity of the cells was measured using the WST-1 cell proliferation assay. In GSU cells, HB-EGF but not AREG and EGF was effective in rescuing the cells from trastuzumab treatment. In H111TC, a similar but not significant trend was observed. No effect of either ligand was detected for the trastuzumab-resistant cell lines KATOIII and MKN45. The mean value of three independent experiments is shown. For better readability only p values referring to the control are shown. For all significant p values, please refer to Table 2 . p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively
    Figure Legend Snippet: Effect of exogenous ligand application on trastuzumab sensitivity in gastric cancer cell lines. GSU, H111TC, KATOIII and MKN45 cells were treated for 3 days with trastuzumab alone (0, 1, 20 µg/ml) and/or different HER receptor ligands (AREG: 15 ng/ml; EGF: 0.1 ng/ml; HB-EGF: 0.4 ng/ml). The metabolic activity of the cells was measured using the WST-1 cell proliferation assay. In GSU cells, HB-EGF but not AREG and EGF was effective in rescuing the cells from trastuzumab treatment. In H111TC, a similar but not significant trend was observed. No effect of either ligand was detected for the trastuzumab-resistant cell lines KATOIII and MKN45. The mean value of three independent experiments is shown. For better readability only p values referring to the control are shown. For all significant p values, please refer to Table 2 . p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively

    Techniques Used: Activity Assay, Proliferation Assay

    Effect of trastuzumab treatment on the cell proliferation of gastric cancer cell lines. The gastric cell lines AGS, GSU, H111TC, HGC-27, Hs746T, KATOIII, LMSU, MKN1, MKN7, MKN28 and MKN45 were treated for 72 h with the indicated amounts of trastuzumab (0/0.1/1/10/20/40 µg/ml), a solvent control (Sol), an isotype control (ISO) or isotype solvent control (ISO-Sol). Afterwards, the metabolic activity of the cell lines was determined via WST-1 cell proliferation assay. Only GSU and H111TC cells were trastuzumab sensitive ( dark grey diagrams ). The mean value of at least three independent experiments is shown. p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively
    Figure Legend Snippet: Effect of trastuzumab treatment on the cell proliferation of gastric cancer cell lines. The gastric cell lines AGS, GSU, H111TC, HGC-27, Hs746T, KATOIII, LMSU, MKN1, MKN7, MKN28 and MKN45 were treated for 72 h with the indicated amounts of trastuzumab (0/0.1/1/10/20/40 µg/ml), a solvent control (Sol), an isotype control (ISO) or isotype solvent control (ISO-Sol). Afterwards, the metabolic activity of the cell lines was determined via WST-1 cell proliferation assay. Only GSU and H111TC cells were trastuzumab sensitive ( dark grey diagrams ). The mean value of at least three independent experiments is shown. p values at significance levels of ≤0.050 and ≤0.010 are indicated by (*) and (**), respectively

    Techniques Used: Activity Assay, Proliferation Assay

    59) Product Images from "Telomere heterogeneity linked to metabolism and pluripotency state revealed by simultaneous analysis of telomere length and RNA-seq in the same human embryonic stem cell"

    Article Title: Telomere heterogeneity linked to metabolism and pluripotency state revealed by simultaneous analysis of telomere length and RNA-seq in the same human embryonic stem cell

    Journal: BMC Biology

    doi: 10.1186/s12915-017-0453-8

    Short telomere human embryonic stem cells (hESCs) display increased oxidative phosphorylation and decreased glycolysis. a Top 14 KEGG pathway annotations of significantly up-regulated genes in short telomere group compared with long telomere group. The terms are sorted by their enrichment P value and the shape indicates the number of differentially expressed genes. b Boxplots illustrating distribution in the expression of genes associated with oxidative phosphorylation and glycolysis in the various telomere length ( L long, M medium, S short) groups. Gene list of oxidative phosphorylation from KEGG and genes related to glycolysis were obtained from Gu et al. [ 49 ]
    Figure Legend Snippet: Short telomere human embryonic stem cells (hESCs) display increased oxidative phosphorylation and decreased glycolysis. a Top 14 KEGG pathway annotations of significantly up-regulated genes in short telomere group compared with long telomere group. The terms are sorted by their enrichment P value and the shape indicates the number of differentially expressed genes. b Boxplots illustrating distribution in the expression of genes associated with oxidative phosphorylation and glycolysis in the various telomere length ( L long, M medium, S short) groups. Gene list of oxidative phosphorylation from KEGG and genes related to glycolysis were obtained from Gu et al. [ 49 ]

    Techniques Used: Expressing

    NANOG and OCT4/POU5F1 expression levels in association with telomere length and pluripotency of human embryonic stem cell (hESCs). a Telomere fluorescence signal (green, TelC-FITC, F1009, Panagene) and NANOG expression (red) in hESCs revealed by IF-FISH. DAPI-stained nuclei. b Linear regression analysis of telomere length and NANOG protein expression at single cell level. c Telomere fluorescence signal (red, TelC-Cy3, F1002, Panagene) and OCT4/POU5F1 expression (green) in hESCs revealed by IF-FISH. DAPI-stained nuclei. d Linear regression analysis of telomere length and OCT4/POU5F1 protein expression at single cell level
    Figure Legend Snippet: NANOG and OCT4/POU5F1 expression levels in association with telomere length and pluripotency of human embryonic stem cell (hESCs). a Telomere fluorescence signal (green, TelC-FITC, F1009, Panagene) and NANOG expression (red) in hESCs revealed by IF-FISH. DAPI-stained nuclei. b Linear regression analysis of telomere length and NANOG protein expression at single cell level. c Telomere fluorescence signal (red, TelC-Cy3, F1002, Panagene) and OCT4/POU5F1 expression (green) in hESCs revealed by IF-FISH. DAPI-stained nuclei. d Linear regression analysis of telomere length and OCT4/POU5F1 protein expression at single cell level

    Techniques Used: Expressing, Fluorescence, Fluorescence In Situ Hybridization, Staining

    TERF1 expression levels associated with telomere length and pluripotency of human embryonic stem cell (hESCs). a Scheme for hTERF1 knockout by CRISPR/Cas9. b Morphology of TERF1 +/- and wild-type (WT) hESCs (WA26) at passage 11. Scale bar = 100 μm. c Expression levels of pluripotency and telomerase genes in TERF1 +/- and WT hESCs by qPCR analysis. * P
    Figure Legend Snippet: TERF1 expression levels associated with telomere length and pluripotency of human embryonic stem cell (hESCs). a Scheme for hTERF1 knockout by CRISPR/Cas9. b Morphology of TERF1 +/- and wild-type (WT) hESCs (WA26) at passage 11. Scale bar = 100 μm. c Expression levels of pluripotency and telomerase genes in TERF1 +/- and WT hESCs by qPCR analysis. * P

    Techniques Used: Expressing, Knock-Out, CRISPR, Real-time Polymerase Chain Reaction

    Simultaneous measurement of telomere length and transcriptome in the same cell. a Overview and design of single-cell telomere length measurement and RNA-sequencing analysis (scT R-seq) in the same cell. b Average telomere length in single cells shown as T/R ratio by scT R-seq is significantly correlated with that of other methods in various human cell lines. Each point represents one cell line, and different colors distinguish between methods. c Relative telomere length of TERC knockout and wild-type hESCs using scT R-seq. d Relative telomere length distribution of all individual human embryonic stem cell (hESC, WA26). e Hierarchical clustering analysis of whole-gene expression similarity (measured by Pearson correlation coefficients) for all hESCs. f Heat-map showing expression of selected marker genes for housekeeping (I), differentiation (II), and pluripotency (III) in hESCs. Each row represents one cell
    Figure Legend Snippet: Simultaneous measurement of telomere length and transcriptome in the same cell. a Overview and design of single-cell telomere length measurement and RNA-sequencing analysis (scT R-seq) in the same cell. b Average telomere length in single cells shown as T/R ratio by scT R-seq is significantly correlated with that of other methods in various human cell lines. Each point represents one cell line, and different colors distinguish between methods. c Relative telomere length of TERC knockout and wild-type hESCs using scT R-seq. d Relative telomere length distribution of all individual human embryonic stem cell (hESC, WA26). e Hierarchical clustering analysis of whole-gene expression similarity (measured by Pearson correlation coefficients) for all hESCs. f Heat-map showing expression of selected marker genes for housekeeping (I), differentiation (II), and pluripotency (III) in hESCs. Each row represents one cell

    Techniques Used: RNA Sequencing Assay, Knock-Out, Expressing, Marker

    Telomere length heterogeneity and candidate genes potentially involved in telomere length maintenance of human embryonic stem cells (hESCs). a hESCs were clustered into three telomere length groups based on T/R ratio by single-cell analysis. We defined the cells with T/R ratio over 1.0 as the “long” telomere group, those with T/R ratio 0.4–1.0 as the “medium” group, and those with T/R ratio under 0.4 as the “short” group. Inset pie figure shows cell number in each telomere length group. Inset density figure indicates distribution of telomere length of hESCs. b Top eight genes with expression patterns remarkably correlated with telomere length (T/R ratio). Red and blue bars show positive and negative correlation with each other. c Scatter plots showing correlation between gene expression level and T/R ratio for selected genes in all samples. The line represents fitted values and shaded band shows 95% confident regions. Correlation value of each gene is shown on the plot
    Figure Legend Snippet: Telomere length heterogeneity and candidate genes potentially involved in telomere length maintenance of human embryonic stem cells (hESCs). a hESCs were clustered into three telomere length groups based on T/R ratio by single-cell analysis. We defined the cells with T/R ratio over 1.0 as the “long” telomere group, those with T/R ratio 0.4–1.0 as the “medium” group, and those with T/R ratio under 0.4 as the “short” group. Inset pie figure shows cell number in each telomere length group. Inset density figure indicates distribution of telomere length of hESCs. b Top eight genes with expression patterns remarkably correlated with telomere length (T/R ratio). Red and blue bars show positive and negative correlation with each other. c Scatter plots showing correlation between gene expression level and T/R ratio for selected genes in all samples. The line represents fitted values and shaded band shows 95% confident regions. Correlation value of each gene is shown on the plot

    Techniques Used: Single-cell Analysis, Expressing

    scT R-seq analysis of heterogeneity in pluripotency gene expression of hESCs. a Distribution of PluriNet score. Gene set for PluriNet score was generated from study in GEO (Chu et al . [ 45 ]). b Correlation between the averaged PluriNet score and telomere length (T/R ratio). hESCs were divided into three groups by Log (PluriNet score) and Log (T/R ratio), Group_1 (Log (T/R ratio) > 0; Log (PluriNet score) > 3.0), Group_2 (Log (T/R ratio) ≥ 0; Log (PluriNet score) ≥ 3.0), and Group_3 (Log (T/R ratio)
    Figure Legend Snippet: scT R-seq analysis of heterogeneity in pluripotency gene expression of hESCs. a Distribution of PluriNet score. Gene set for PluriNet score was generated from study in GEO (Chu et al . [ 45 ]). b Correlation between the averaged PluriNet score and telomere length (T/R ratio). hESCs were divided into three groups by Log (PluriNet score) and Log (T/R ratio), Group_1 (Log (T/R ratio) > 0; Log (PluriNet score) > 3.0), Group_2 (Log (T/R ratio) ≥ 0; Log (PluriNet score) ≥ 3.0), and Group_3 (Log (T/R ratio)

    Techniques Used: Expressing, Generated

    Classification of telomere lengths in association with global transcriptome of human embryonic stem cell (hESCs). a Classification of all single cells based on whole transcriptome profile using principal component analysis (PCA, scatter plot of component 1 and 2), with cells colored according to telomere length group. b Venn diagram depicting the overlap among three telomere groups. c Heatmap representing expression levels of 54 selected genes related to telomere length maintenance from GESA gene sets ( http://software.broadinstitute.org/gsea ). “High” means gene expression at high levels; “Low” means low expression levels; “Vary” indicates gene expression levels in a wide range. d Violin plots showing relative expression levels of genes associated with shelterin complex in each telomere length group ( L long, M medium, S short). Y-axis indicates Log2 (TPM + 1)
    Figure Legend Snippet: Classification of telomere lengths in association with global transcriptome of human embryonic stem cell (hESCs). a Classification of all single cells based on whole transcriptome profile using principal component analysis (PCA, scatter plot of component 1 and 2), with cells colored according to telomere length group. b Venn diagram depicting the overlap among three telomere groups. c Heatmap representing expression levels of 54 selected genes related to telomere length maintenance from GESA gene sets ( http://software.broadinstitute.org/gsea ). “High” means gene expression at high levels; “Low” means low expression levels; “Vary” indicates gene expression levels in a wide range. d Violin plots showing relative expression levels of genes associated with shelterin complex in each telomere length group ( L long, M medium, S short). Y-axis indicates Log2 (TPM + 1)

    Techniques Used: Expressing, Software

    60) Product Images from "Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells—Brief Report"

    Article Title: Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells—Brief Report

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.118.311252

    Increased thymus weight in males with depletion of the AR (androgen receptor) in epithelial cells (E-ARKO [epithelial cell-specific AR knockout]). A , Gating strategy for sorting thymic epithelial cells (TECs). B , Assessment of AR knockout by measurement of exon 2 genomic DNA in control (K5-Cre + ) and E-ARKO mice, in enriched CD3 + T cells from thymus (control, n=10; and E-ARKO, n=10) and sorted TECs (control, n=4; and E-ARKO, n=4). * P
    Figure Legend Snippet: Increased thymus weight in males with depletion of the AR (androgen receptor) in epithelial cells (E-ARKO [epithelial cell-specific AR knockout]). A , Gating strategy for sorting thymic epithelial cells (TECs). B , Assessment of AR knockout by measurement of exon 2 genomic DNA in control (K5-Cre + ) and E-ARKO mice, in enriched CD3 + T cells from thymus (control, n=10; and E-ARKO, n=10) and sorted TECs (control, n=4; and E-ARKO, n=4). * P

    Techniques Used: Knock-Out, Mouse Assay

    61) Product Images from "Assessment of genetically engineered Trabulsiella odontotermitis as a ‘Trojan Horse’ for paratransgenesis in termites"

    Article Title: Assessment of genetically engineered Trabulsiella odontotermitis as a ‘Trojan Horse’ for paratransgenesis in termites

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0822-4

    Number of T. odontotermitis -pGFP cells recovered from the gut of the termites of three different colonies after feeding for 2 days on cellulose discs containing T. odontotermitis -pGFP. The arrow indicates the day when the termites were moved to a sterile diet. The experiment had three replicates for each colony and 200 worker and 20 soldier termites were used for each replicate. Error bars indicate Standard Error of Mean (SEM)
    Figure Legend Snippet: Number of T. odontotermitis -pGFP cells recovered from the gut of the termites of three different colonies after feeding for 2 days on cellulose discs containing T. odontotermitis -pGFP. The arrow indicates the day when the termites were moved to a sterile diet. The experiment had three replicates for each colony and 200 worker and 20 soldier termites were used for each replicate. Error bars indicate Standard Error of Mean (SEM)

    Techniques Used:

    Number of Kanamycin resistant T. odontotermitis -Km r :: Tn7 recovered from the gut of the termites of three different colonies. The arrow on the X-axis indicates the day when termites were moved to a sterile diet. The experiment had three replicates for each colony and 200 worker and 20 soldier termites were used for each replicate. Error bars indicate Standard Error of Mean (SEM)
    Figure Legend Snippet: Number of Kanamycin resistant T. odontotermitis -Km r :: Tn7 recovered from the gut of the termites of three different colonies. The arrow on the X-axis indicates the day when termites were moved to a sterile diet. The experiment had three replicates for each colony and 200 worker and 20 soldier termites were used for each replicate. Error bars indicate Standard Error of Mean (SEM)

    Techniques Used:

    a Integration of kanR gene in the chromosome of T. odontotermitis using a Tn7 transposon integration; glmS_F and pstS_R show the position and direction of primers used to confirm the integration b PCR based confirmation of integration of kanR gene in the T. odontotermitis chromosome using glmS_F and pstS_R primers. Tra:: Tn7:: km 1,2,3 are the three different isolates after a Tn7 transposition, control is the wild type T. odontotermitis
    Figure Legend Snippet: a Integration of kanR gene in the chromosome of T. odontotermitis using a Tn7 transposon integration; glmS_F and pstS_R show the position and direction of primers used to confirm the integration b PCR based confirmation of integration of kanR gene in the T. odontotermitis chromosome using glmS_F and pstS_R primers. Tra:: Tn7:: km 1,2,3 are the three different isolates after a Tn7 transposition, control is the wild type T. odontotermitis

    Techniques Used: Polymerase Chain Reaction

    Termite hindgut observed under a Leica DM RXA2 fluorescent microscope after feeding on diet containing T. odontotermitis -pGFP. a 5× Differential interference contrast (DIC), white arrows pointing at termite gut protozoa. b 5× fluorescent, T. odontotermitis -pGFP seen concentrated at the hindgut wall. c Overlay of A and B, T. odontotermitis -pGFP seen in the close vicinity of gut protozoa. d 100× DIC, magnified image of the termite hindgut wall. e 100× fluorescent, magnified image of the termite hindgut wall showing T. odontotermitis -pGFP cells expressing GFP. f Overlay of d and e
    Figure Legend Snippet: Termite hindgut observed under a Leica DM RXA2 fluorescent microscope after feeding on diet containing T. odontotermitis -pGFP. a 5× Differential interference contrast (DIC), white arrows pointing at termite gut protozoa. b 5× fluorescent, T. odontotermitis -pGFP seen concentrated at the hindgut wall. c Overlay of A and B, T. odontotermitis -pGFP seen in the close vicinity of gut protozoa. d 100× DIC, magnified image of the termite hindgut wall. e 100× fluorescent, magnified image of the termite hindgut wall showing T. odontotermitis -pGFP cells expressing GFP. f Overlay of d and e

    Techniques Used: Microscopy, Expressing

    T. odontotermitis -Km r :: Tn7 recovered from the gut of the recipient termites (donor: recipient ratio 1:1) of three different colonies. The experiment had three replicates for each colony. Error bars indicate Standard Error of Mean (SEM)
    Figure Legend Snippet: T. odontotermitis -Km r :: Tn7 recovered from the gut of the recipient termites (donor: recipient ratio 1:1) of three different colonies. The experiment had three replicates for each colony. Error bars indicate Standard Error of Mean (SEM)

    Techniques Used:

    62) Product Images from "Lentiviral vectors can be used for full-length dystrophin gene therapy"

    Article Title: Lentiviral vectors can be used for full-length dystrophin gene therapy

    Journal: Scientific Reports

    doi: 10.1038/srep44775

    Packaging of full-length dystrophin into a standard lentiviral expression vector. ( a ) Schematic representing the CCL-SFFV-FLAG-Dystrophin-P2A-GFP expression cassette. The locations of primers used for provirus amplification are marked with angled red lines. ( b ) CCL-SFFV-Dystrophin-P2A-GFP was titred by GFP output after HEK 293T transduction. CCL-SFFV-GFP (CCL-GFP) was titred simultaneously to estimate titre-loss from dystrophin payload packaging. This comparison showed that a functional titre > 1 × 10 6 lp/ml can be obtained from a lentivirus containing full-length dystrophin, which is 2 orders of magnitude lower than the CCL-GFP vector. Bars represent mean log titres with standard deviation from the mean. N = 3 for both samples. ( c ) PCR of CCL-SFFV-Dystrophin-P2A-GFP provirus from GFP-sorted HEK 293T genomic DNA. Running samples on a 1% agarose gel reveals a band of more than 10,000 base pairs in the GFP-sorted sample, which is absent from the untreated control. The expected band size for a provirus containing full-length dystrophin is 14,750 base-pairs.
    Figure Legend Snippet: Packaging of full-length dystrophin into a standard lentiviral expression vector. ( a ) Schematic representing the CCL-SFFV-FLAG-Dystrophin-P2A-GFP expression cassette. The locations of primers used for provirus amplification are marked with angled red lines. ( b ) CCL-SFFV-Dystrophin-P2A-GFP was titred by GFP output after HEK 293T transduction. CCL-SFFV-GFP (CCL-GFP) was titred simultaneously to estimate titre-loss from dystrophin payload packaging. This comparison showed that a functional titre > 1 × 10 6 lp/ml can be obtained from a lentivirus containing full-length dystrophin, which is 2 orders of magnitude lower than the CCL-GFP vector. Bars represent mean log titres with standard deviation from the mean. N = 3 for both samples. ( c ) PCR of CCL-SFFV-Dystrophin-P2A-GFP provirus from GFP-sorted HEK 293T genomic DNA. Running samples on a 1% agarose gel reveals a band of more than 10,000 base pairs in the GFP-sorted sample, which is absent from the untreated control. The expected band size for a provirus containing full-length dystrophin is 14,750 base-pairs.

    Techniques Used: Expressing, Plasmid Preparation, Amplification, Transduction, Functional Assay, Standard Deviation, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    63) Product Images from "Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells"

    Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells

    Journal: eLife

    doi: 10.7554/eLife.36187

    GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.
    Figure Legend Snippet: GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.

    Techniques Used: FACS, Mouse Assay, RNA Sequencing Assay, Sequencing, Expressing, Purification

    64) Product Images from "Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells"

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1152

    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P
    Figure Legend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    65) Product Images from "Impact of trehalose transporter knockdown on Anopheles gambiae stress adaptation and susceptibility to Plasmodium falciparum infection"

    Article Title: Impact of trehalose transporter knockdown on Anopheles gambiae stress adaptation and susceptibility to Plasmodium falciparum infection

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

    doi: 10.1073/pnas.1316709110

    Ag TreT1 silencing affects hemolymph trehalose levels. ( A ) Ag TreT1 mRNA levels reduced in Ag TreT1 dsRNA-injected mosquitoes to as low as 16% of controls during 6 d after injection. RNA of whole mosquitoes was used for reverse transcription and quantitative PCR, and data were normalized using the expression of S7 ribosomal gene as internal control. Black bars represent normalized mRNA level of mosquitoes injected with Ag TreT1 dsRNA, and gray bars represent mosquitoes injected with GFP dsRNA as controls. ( Inset ) Western blot of whole female A. gambiae injected with GFP RNAi or Ag TreT1 RNAi on day 0 (before injection) and day 2, 4, and 9 postinjection. ( Upper ) Blot against anti- Ag TreT1 antibody. ( Lower ) Blot against anti-Actin antibody. ( B ) The relative concentration of hemolymph trehalose in Ag TreT1 or GFP dsRNA-injected A. gambiae mosquitoes 8 d after injection. Trehalose concentrations were normalized to genomic DNA extracted from hemolymph cells. Data represent mean ± SD, * P ≤ 0.05 by Student's t test.
    Figure Legend Snippet: Ag TreT1 silencing affects hemolymph trehalose levels. ( A ) Ag TreT1 mRNA levels reduced in Ag TreT1 dsRNA-injected mosquitoes to as low as 16% of controls during 6 d after injection. RNA of whole mosquitoes was used for reverse transcription and quantitative PCR, and data were normalized using the expression of S7 ribosomal gene as internal control. Black bars represent normalized mRNA level of mosquitoes injected with Ag TreT1 dsRNA, and gray bars represent mosquitoes injected with GFP dsRNA as controls. ( Inset ) Western blot of whole female A. gambiae injected with GFP RNAi or Ag TreT1 RNAi on day 0 (before injection) and day 2, 4, and 9 postinjection. ( Upper ) Blot against anti- Ag TreT1 antibody. ( Lower ) Blot against anti-Actin antibody. ( B ) The relative concentration of hemolymph trehalose in Ag TreT1 or GFP dsRNA-injected A. gambiae mosquitoes 8 d after injection. Trehalose concentrations were normalized to genomic DNA extracted from hemolymph cells. Data represent mean ± SD, * P ≤ 0.05 by Student's t test.

    Techniques Used: Injection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay

    66) Product Images from "The ROP16III-dependent early immune response determines the subacute CNS immune response and type III Toxoplasma gondii survival"

    Article Title: The ROP16III-dependent early immune response determines the subacute CNS immune response and type III Toxoplasma gondii survival

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007856

    Type II and type III-infected mice show similar dissemination pattern and parasite burdens in early and subacute CNS infection. Mice were inoculated with type II or type III parasites, and brains were harvested at noted time points. A. Quantification of CNS Toxoplasma burden at 21 dpi using quantitative PCR (qPCR) for the Toxoplasma -specific B1 gene and host GAPDH gene (housekeeping gene). Toxoplasma and mouse genomic DNA were isolated from brain homogenates. B. Quantification of Toxoplasma cyst burden at 21 dpi in brain sections stained with Dolichos biflorous agglutinin (DBA), which stains the cyst wall. Stained sections were then analyzed by epifluorescent microscopy to quantify DBA + mCherry + cysts. For A,B . Bars, mean ± SEM. N = 8 mice/infected group. ns = not significant; two-way ANOVA with Fisher’s protected LSD. Data representative of 3 independent experiments. C,D,E,F. Quantification of Toxoplasma burden was performed as in ( A ) at specified time points from spleen, liver, lung, and brain. Bars, mean ± SEM. N = 4–5 mice/infected group/time point. No significant differences were found in mean B1 quantification. Two-way ANOVA with Fisher’s protected LSD. Data are representative of 2 independent experiments. ● = type II, ■ = type III.
    Figure Legend Snippet: Type II and type III-infected mice show similar dissemination pattern and parasite burdens in early and subacute CNS infection. Mice were inoculated with type II or type III parasites, and brains were harvested at noted time points. A. Quantification of CNS Toxoplasma burden at 21 dpi using quantitative PCR (qPCR) for the Toxoplasma -specific B1 gene and host GAPDH gene (housekeeping gene). Toxoplasma and mouse genomic DNA were isolated from brain homogenates. B. Quantification of Toxoplasma cyst burden at 21 dpi in brain sections stained with Dolichos biflorous agglutinin (DBA), which stains the cyst wall. Stained sections were then analyzed by epifluorescent microscopy to quantify DBA + mCherry + cysts. For A,B . Bars, mean ± SEM. N = 8 mice/infected group. ns = not significant; two-way ANOVA with Fisher’s protected LSD. Data representative of 3 independent experiments. C,D,E,F. Quantification of Toxoplasma burden was performed as in ( A ) at specified time points from spleen, liver, lung, and brain. Bars, mean ± SEM. N = 4–5 mice/infected group/time point. No significant differences were found in mean B1 quantification. Two-way ANOVA with Fisher’s protected LSD. Data are representative of 2 independent experiments. ● = type II, ■ = type III.

    Techniques Used: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Staining, Microscopy

    67) Product Images from "The iCRISPR platform for rapid genome editing in human Pluripotent Stem Cells"

    Article Title: The iCRISPR platform for rapid genome editing in human Pluripotent Stem Cells

    Journal: Methods in enzymology

    doi: 10.1016/B978-0-12-801185-0.00011-8

    Southern blot genotyping of AAVS1 -targeted hPSC iCas9 lines DNA is digested with either BgIII ( B ), for 3′ external probe hybridization, or SphI ( S ), for 5′ internal probe hybridization. BglII digestion and 3′ external probe hybridization yield bands of 12406, 7409 and 4984 bp for wild-type, Neo-M2rtTA, Puro-iCas9 (or Puro-iCr); whereas SphI digestion and 5′ internal probe hybridization yields bands of 6492, 3492 and 3781 bp respectively.]
    Figure Legend Snippet: Southern blot genotyping of AAVS1 -targeted hPSC iCas9 lines DNA is digested with either BgIII ( B ), for 3′ external probe hybridization, or SphI ( S ), for 5′ internal probe hybridization. BglII digestion and 3′ external probe hybridization yield bands of 12406, 7409 and 4984 bp for wild-type, Neo-M2rtTA, Puro-iCas9 (or Puro-iCr); whereas SphI digestion and 5′ internal probe hybridization yields bands of 6492, 3492 and 3781 bp respectively.]

    Techniques Used: Southern Blot, Hybridization

    The iCRISPR platform for rapid genome editing in hPSCs Instead of transient expression of Cas9 and gRNAs from electroporated plasmids in hPSCs, targeted integration and inducible expression of Cas9 into the AAVS1 locus provides a precise and reliable approach to express the invariable component of the CRISPR/Cas system (Generation of iCas9 hPSCs, Section 2). Then, gRNAs targeting specific loci are designed and synthesized by PCR amplification of in vitro transcription DNA templates (gRNA production, Section 3.1 and 3.2). Due to their small size (∼100 nucleotides), transfection of iCas9 hPSCs with gRNAs is highly efficient, thus could lead to reproducible and highly efficient gene knockout in hPSCs (Generation of hPSC knockouts by gRNA transfection, Section 3.3).]
    Figure Legend Snippet: The iCRISPR platform for rapid genome editing in hPSCs Instead of transient expression of Cas9 and gRNAs from electroporated plasmids in hPSCs, targeted integration and inducible expression of Cas9 into the AAVS1 locus provides a precise and reliable approach to express the invariable component of the CRISPR/Cas system (Generation of iCas9 hPSCs, Section 2). Then, gRNAs targeting specific loci are designed and synthesized by PCR amplification of in vitro transcription DNA templates (gRNA production, Section 3.1 and 3.2). Due to their small size (∼100 nucleotides), transfection of iCas9 hPSCs with gRNAs is highly efficient, thus could lead to reproducible and highly efficient gene knockout in hPSCs (Generation of hPSC knockouts by gRNA transfection, Section 3.3).]

    Techniques Used: Expressing, CRISPR, Synthesized, Polymerase Chain Reaction, Amplification, In Vitro, Transfection, Gene Knockout

    68) Product Images from "The iCRISPR platform for rapid genome editing in human Pluripotent Stem Cells"

    Article Title: The iCRISPR platform for rapid genome editing in human Pluripotent Stem Cells

    Journal: Methods in enzymology

    doi: 10.1016/B978-0-12-801185-0.00011-8

    Inducible gene knockout in hPSCs using iCRISPR (A) iCas9 hPSCs are first differentiation into required cell types. Induced expression of Cas9 with doxycycline treatment and gRNA transfection in differentiated cells result in inducible gene knockout. (B) In iCr hPSCs, a constitutive gRNA expression module is inserted 3′ of the Cas9 expression cassette. Treat differentiated iCr hPSCs with doxycycline induces Cas9 expression thus induces gene knockout. ]
    Figure Legend Snippet: Inducible gene knockout in hPSCs using iCRISPR (A) iCas9 hPSCs are first differentiation into required cell types. Induced expression of Cas9 with doxycycline treatment and gRNA transfection in differentiated cells result in inducible gene knockout. (B) In iCr hPSCs, a constitutive gRNA expression module is inserted 3′ of the Cas9 expression cassette. Treat differentiated iCr hPSCs with doxycycline induces Cas9 expression thus induces gene knockout. ]

    Techniques Used: Gene Knockout, Expressing, Transfection

    The iCRISPR platform for rapid genome editing in hPSCs Instead of transient expression of Cas9 and gRNAs from electroporated plasmids in hPSCs, targeted integration and inducible expression of Cas9 into the AAVS1 locus provides a precise and reliable approach to express the invariable component of the CRISPR/Cas system (Generation of iCas9 hPSCs, Section 2). Then, gRNAs targeting specific loci are designed and synthesized by PCR amplification of in vitro transcription DNA templates (gRNA production, Section 3.1 and 3.2). Due to their small size (∼100 nucleotides), transfection of iCas9 hPSCs with gRNAs is highly efficient, thus could lead to reproducible and highly efficient gene knockout in hPSCs (Generation of hPSC knockouts by gRNA transfection, Section 3.3).]
    Figure Legend Snippet: The iCRISPR platform for rapid genome editing in hPSCs Instead of transient expression of Cas9 and gRNAs from electroporated plasmids in hPSCs, targeted integration and inducible expression of Cas9 into the AAVS1 locus provides a precise and reliable approach to express the invariable component of the CRISPR/Cas system (Generation of iCas9 hPSCs, Section 2). Then, gRNAs targeting specific loci are designed and synthesized by PCR amplification of in vitro transcription DNA templates (gRNA production, Section 3.1 and 3.2). Due to their small size (∼100 nucleotides), transfection of iCas9 hPSCs with gRNAs is highly efficient, thus could lead to reproducible and highly efficient gene knockout in hPSCs (Generation of hPSC knockouts by gRNA transfection, Section 3.3).]

    Techniques Used: Expressing, CRISPR, Synthesized, Polymerase Chain Reaction, Amplification, In Vitro, Transfection, Gene Knockout

    69) Product Images from "Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion"

    Article Title: Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

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

    doi: 10.1073/pnas.1522223113

    Inhibition of the oxidative pentose-phosphate pathway with 6AN does not synergize with Olaparib treatments. The effects of 6AN alone or in combination with the Parp-inhibitor Olaparib were examined in K14-Cre BRCA1 f/f p53 f/f breast cancer cells ( A and C ) or HCC1937 human breast cancer cells ( B and D ). ( A and B ) Cell growth as determined by CellTiter-Glo assay over time (in days). ( C and D ) Cells were treated for 16 h with drugs as indicated and subjected to immunoblotting with antibodies as indicated.
    Figure Legend Snippet: Inhibition of the oxidative pentose-phosphate pathway with 6AN does not synergize with Olaparib treatments. The effects of 6AN alone or in combination with the Parp-inhibitor Olaparib were examined in K14-Cre BRCA1 f/f p53 f/f breast cancer cells ( A and C ) or HCC1937 human breast cancer cells ( B and D ). ( A and B ) Cell growth as determined by CellTiter-Glo assay over time (in days). ( C and D ) Cells were treated for 16 h with drugs as indicated and subjected to immunoblotting with antibodies as indicated.

    Techniques Used: Inhibition, Glo Assay

    PI3K, but not AKT, MAPKK, or SGK inhibition induces markers of DNA damage in BRCA1- mutant breast cancers. For A – C , HCC1937 cells were treated for 16 h with inhibitors as indicated. Immunoblotting of total cell lysates was performed with antibodies as indicated. ( A ) Induction of PAR and H2ax phosphorylation (γH2ax) following treatment with inhibitors of pan-PI3K (BKM120, 1.5 μM), PI3Kα (BYL719, 3 μM; PIK75, 0.5 μM), PI3Kβ (TGX221, 30 μM), the PARP-inhibitor Olaparib (5 μM), and inhibitors of AKT (MK2206, 1 μM), SGK (GSK650394, 10 μM), or MAPKK (GSK1120212, 5 nM). ( B ) Induction of PAR and γH2ax by the PIP3-mimetic PIT1. ( C ) Neither AKT nor SGK, nor the combination of AKT and SGK inhibitors, induces PAR or γH2ax. ( D and E ) Induction of DNA damage indicators in vivo. K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice were treated with two doses of drugs as indicated [BKM120 30 mg/kg by mouth (PO), BYL719 30 mg/kg PO, Olaparib 50 mg/kg i.p.] 8 and 2 h before killing. Tumors were immediately harvested and processed for immunoblotting of fresh tumor tissue lysates with antibodies as indicated ( D ) or fixed and stained for immunofluorescence with antibodies as indicated ( E ). Insets (400× magnification) show representative single cells stained for pATM ( Upper ) or pRPA ( Lower ).
    Figure Legend Snippet: PI3K, but not AKT, MAPKK, or SGK inhibition induces markers of DNA damage in BRCA1- mutant breast cancers. For A – C , HCC1937 cells were treated for 16 h with inhibitors as indicated. Immunoblotting of total cell lysates was performed with antibodies as indicated. ( A ) Induction of PAR and H2ax phosphorylation (γH2ax) following treatment with inhibitors of pan-PI3K (BKM120, 1.5 μM), PI3Kα (BYL719, 3 μM; PIK75, 0.5 μM), PI3Kβ (TGX221, 30 μM), the PARP-inhibitor Olaparib (5 μM), and inhibitors of AKT (MK2206, 1 μM), SGK (GSK650394, 10 μM), or MAPKK (GSK1120212, 5 nM). ( B ) Induction of PAR and γH2ax by the PIP3-mimetic PIT1. ( C ) Neither AKT nor SGK, nor the combination of AKT and SGK inhibitors, induces PAR or γH2ax. ( D and E ) Induction of DNA damage indicators in vivo. K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice were treated with two doses of drugs as indicated [BKM120 30 mg/kg by mouth (PO), BYL719 30 mg/kg PO, Olaparib 50 mg/kg i.p.] 8 and 2 h before killing. Tumors were immediately harvested and processed for immunoblotting of fresh tumor tissue lysates with antibodies as indicated ( D ) or fixed and stained for immunofluorescence with antibodies as indicated ( E ). Insets (400× magnification) show representative single cells stained for pATM ( Upper ) or pRPA ( Lower ).

    Techniques Used: Inhibition, Mutagenesis, In Vivo, Mouse Assay, Staining, Immunofluorescence

    Response to PI3K and PARP inhibition in vivo. Six different primary tumors from K14-Cre BRCA1 f/f p53 f/f females were propagated in vivo through syngeneic transplantation. Recipient females were randomized to treatments with BKM120 (30 mg⋅kg⋅ −1 d −1 by mouth), BYL719 (30 mg⋅kg⋅ −1 ⋅d −1 by mouth), Olaparib (50 mg⋅kg⋅ −1 d −1 i.p.), or their combination, and tumor volume was recorded every 2 d. Treatment endpoint was time to progression as defined by tumor volume doubling. ( A ) Two mice per treatment condition were killed after 8 and 72 h of drug treatments to assess biomarkers of response, as assessed with Ki67 and CC3 by IHC (magnification: 200×). These mice were given a last dose of drugs 2 h before killing. ( B ) Survival statistics (log-rank test) and Kaplan–Meier analysis of 132 recipient females randomized to treatments as indicated. ( C ) Resistance to PI3K inhibition. 18 FDG-PET-CT scan at baseline and after 48 h of administration of the PI3K inhibitor NVP-BKM120 (3 doses, 30 mg/kg PO) in sensitive and BKM120+Olaparib resistant K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice. The maximum standardized uptake value (SUVmax) of tumors before and after PI3K inhibitor administration is displayed in the bar graph. Tumors are marked with a yellow arrow. Two hours before killing, mice were injected with BrdU and given an additional dose of BKM120. Tumor sections were stained with anti-BrdU antibodies for immunofluoresence (red) (magnification: 200×) and mean fluorescence per cell of BrdU + cells was measured using volocity software. ( D ) A cell line was derived from the clinically resistant tumor and its PI3K/PARP-sensitive parental tumor. Cells were treated in vitro with drugs as indicated for 16 h, lysed, and blotted with antibodies as indicated.
    Figure Legend Snippet: Response to PI3K and PARP inhibition in vivo. Six different primary tumors from K14-Cre BRCA1 f/f p53 f/f females were propagated in vivo through syngeneic transplantation. Recipient females were randomized to treatments with BKM120 (30 mg⋅kg⋅ −1 d −1 by mouth), BYL719 (30 mg⋅kg⋅ −1 ⋅d −1 by mouth), Olaparib (50 mg⋅kg⋅ −1 d −1 i.p.), or their combination, and tumor volume was recorded every 2 d. Treatment endpoint was time to progression as defined by tumor volume doubling. ( A ) Two mice per treatment condition were killed after 8 and 72 h of drug treatments to assess biomarkers of response, as assessed with Ki67 and CC3 by IHC (magnification: 200×). These mice were given a last dose of drugs 2 h before killing. ( B ) Survival statistics (log-rank test) and Kaplan–Meier analysis of 132 recipient females randomized to treatments as indicated. ( C ) Resistance to PI3K inhibition. 18 FDG-PET-CT scan at baseline and after 48 h of administration of the PI3K inhibitor NVP-BKM120 (3 doses, 30 mg/kg PO) in sensitive and BKM120+Olaparib resistant K14-Cre BRCA1 f/f p53 f/f tumor-bearing mice. The maximum standardized uptake value (SUVmax) of tumors before and after PI3K inhibitor administration is displayed in the bar graph. Tumors are marked with a yellow arrow. Two hours before killing, mice were injected with BrdU and given an additional dose of BKM120. Tumor sections were stained with anti-BrdU antibodies for immunofluoresence (red) (magnification: 200×) and mean fluorescence per cell of BrdU + cells was measured using volocity software. ( D ) A cell line was derived from the clinically resistant tumor and its PI3K/PARP-sensitive parental tumor. Cells were treated in vitro with drugs as indicated for 16 h, lysed, and blotted with antibodies as indicated.

    Techniques Used: Inhibition, In Vivo, Transplantation Assay, Mouse Assay, Immunohistochemistry, Positron Emission Tomography, Computed Tomography, Injection, Staining, Fluorescence, Software, Derivative Assay, In Vitro

    Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into cell biomass ( A ) or DNA ( B and C ) in response to PI3K and PARP inhibition. HCC1937 cells were cultured in the presence of 14 C 6 -glucose or 14 C 1 -glucose, BKM120 (1 μM) or Olaparib (5 μM), or their combination for 3 h as indicated. Scintillation was counted for the entire cell lysate ( A ); genomic DNA was extracted and 14 C measured ( B and C ). ( D ) Loss of PI3Kα leads to induction of H2AX phosphorylation (γH2AX) that can be rescued by nucleoside reconstitution. HCC1937 cells were depleted of PI3Kα by using siRNA for 48 h, and then treated with or without nucleosides for another 16 h. Cell lysates were subjected to immunoblotting with antibodies as indicated. ( E ) A radioactive label in the 6 position will measure flux through both oxidative and nonoxidative PPP, whereas a label in the 1 position will allow to measure flux through the nonoxidative PPP only.
    Figure Legend Snippet: Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into cell biomass ( A ) or DNA ( B and C ) in response to PI3K and PARP inhibition. HCC1937 cells were cultured in the presence of 14 C 6 -glucose or 14 C 1 -glucose, BKM120 (1 μM) or Olaparib (5 μM), or their combination for 3 h as indicated. Scintillation was counted for the entire cell lysate ( A ); genomic DNA was extracted and 14 C measured ( B and C ). ( D ) Loss of PI3Kα leads to induction of H2AX phosphorylation (γH2AX) that can be rescued by nucleoside reconstitution. HCC1937 cells were depleted of PI3Kα by using siRNA for 48 h, and then treated with or without nucleosides for another 16 h. Cell lysates were subjected to immunoblotting with antibodies as indicated. ( E ) A radioactive label in the 6 position will measure flux through both oxidative and nonoxidative PPP, whereas a label in the 1 position will allow to measure flux through the nonoxidative PPP only.

    Techniques Used: Derivative Assay, Inhibition, Cell Culture

    PI3K inhibition leads to decreased glycolytic flux through the nonoxidative pentose phosphate pathway. ( A ) Seahorse assay to determine overall glycolytic flux. Cells were seeded at 5,000 cells per well in a 24-well plate the night before the assay. Drugs were added at 3 h. ECAR was measured every minute for a total of 120 min in response to a glucose challenge, mitochondrial uncoupling to mobilize glycolytic reserve with oligomycin, and disruption of glycolysis with 2DG. Displayed is the ECAR over time of experimental quadruplicates ± SD. ( B ) Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into DNA in response to PI3K and PARP inhibition. Cells were treated with 1- 14 C- or 6- 14 C-glucose and drugs as indicated 8 h before lysis. Scintillation count was done on purified genomic DNA. Displayed is the 14 C-uptake of experimental triplicates ± SD, normalized to 14 C-uptake in the 6- 14 C-glucose control. ( C ) Effect of PI3K and AKT inhibition on Rib-phosphate synthesis in HCC1937 cells. Cells were treated with vehicle control, BKM120 (1 μM), or MK2206 (1 μM) for 3 h, followed by labeling with [U- 13 C 6 ]-glucose for 60 s and processed for mass spectrometry. ( D ) Nucleoside rescue of PI3K inhibitor-induced DNA damage indicators. Cells were treated for 16 h with drugs as indicated in the presence of a mixture of four nucleosides. ( E ) Determination of nucleotide levels in HCC1937 cells after 16 h of drug treatment with either BKM120 or MK2206 in the presence or absence of nucleosides. Determinations were done by competitive PCR, and displayed are the results of experimental triplicates. ( F ) Nucleoside rescue of PI3K inhibitor-induced decrease in DNA synthesis. Cells were treated for 8 h with drugs (BYL719, 2.5 μM; BKM120, 1 μM; or Olaparib, 5 μM) as indicated in the presence or absence of a mixture of four nucleosides. EdU was added in the last 2 h. Displayed are mean fluorescence of the EdU + population relative to vehicle control in experimental triplicates. Significance was P
    Figure Legend Snippet: PI3K inhibition leads to decreased glycolytic flux through the nonoxidative pentose phosphate pathway. ( A ) Seahorse assay to determine overall glycolytic flux. Cells were seeded at 5,000 cells per well in a 24-well plate the night before the assay. Drugs were added at 3 h. ECAR was measured every minute for a total of 120 min in response to a glucose challenge, mitochondrial uncoupling to mobilize glycolytic reserve with oligomycin, and disruption of glycolysis with 2DG. Displayed is the ECAR over time of experimental quadruplicates ± SD. ( B ) Carbon flux from glucose to Rib as determined by 14 C-glucose–derived carbon into DNA in response to PI3K and PARP inhibition. Cells were treated with 1- 14 C- or 6- 14 C-glucose and drugs as indicated 8 h before lysis. Scintillation count was done on purified genomic DNA. Displayed is the 14 C-uptake of experimental triplicates ± SD, normalized to 14 C-uptake in the 6- 14 C-glucose control. ( C ) Effect of PI3K and AKT inhibition on Rib-phosphate synthesis in HCC1937 cells. Cells were treated with vehicle control, BKM120 (1 μM), or MK2206 (1 μM) for 3 h, followed by labeling with [U- 13 C 6 ]-glucose for 60 s and processed for mass spectrometry. ( D ) Nucleoside rescue of PI3K inhibitor-induced DNA damage indicators. Cells were treated for 16 h with drugs as indicated in the presence of a mixture of four nucleosides. ( E ) Determination of nucleotide levels in HCC1937 cells after 16 h of drug treatment with either BKM120 or MK2206 in the presence or absence of nucleosides. Determinations were done by competitive PCR, and displayed are the results of experimental triplicates. ( F ) Nucleoside rescue of PI3K inhibitor-induced decrease in DNA synthesis. Cells were treated for 8 h with drugs (BYL719, 2.5 μM; BKM120, 1 μM; or Olaparib, 5 μM) as indicated in the presence or absence of a mixture of four nucleosides. EdU was added in the last 2 h. Displayed are mean fluorescence of the EdU + population relative to vehicle control in experimental triplicates. Significance was P

    Techniques Used: Inhibition, Derivative Assay, Lysis, Purification, Labeling, Mass Spectrometry, Polymerase Chain Reaction, DNA Synthesis, Fluorescence

    70) Product Images from "HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site"

    Article Title: HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.01135-18

    Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.
    Figure Legend Snippet: Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.

    Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification, Expressing, Clone Assay, Variant Assay

    Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.
    Figure Legend Snippet: Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.

    Techniques Used: Inhibition, Produced, Expressing, Activity Assay

    The PBS-M1 and PBS-M2 mutations allow HIV-1 to resist Cas9/sgPBS1. (A) Depiction of the M1 and M2 mutations (in red letters) in the PBS. (B) M1 and M2 mutations resist Cas9/sgPBS1 inhibition in short-term infection. Wild-type HIV-1 or the M1 or M2 mutant, with the same p24 amounts, was used to infect SupT1 cells expressing the Cas9 clone (control) or Cas9/sgPBS1. Forty hours after infection, the levels of HIV-1 in the culture supernatants were determined by infecting the TZM-bl indicator cells. Results shown are the average values from three independent infection experiments. Levels of each virus from the control cells are set at 100. ***, P
    Figure Legend Snippet: The PBS-M1 and PBS-M2 mutations allow HIV-1 to resist Cas9/sgPBS1. (A) Depiction of the M1 and M2 mutations (in red letters) in the PBS. (B) M1 and M2 mutations resist Cas9/sgPBS1 inhibition in short-term infection. Wild-type HIV-1 or the M1 or M2 mutant, with the same p24 amounts, was used to infect SupT1 cells expressing the Cas9 clone (control) or Cas9/sgPBS1. Forty hours after infection, the levels of HIV-1 in the culture supernatants were determined by infecting the TZM-bl indicator cells. Results shown are the average values from three independent infection experiments. Levels of each virus from the control cells are set at 100. ***, P

    Techniques Used: Inhibition, Infection, Mutagenesis, Expressing

    Development of HIV-1 resistance to Cas9/sgPBS1. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS1-expressing SupT1 cells were harvested. Viral RNA was extracted and subjected to RT-PCR and sequencing. Each indel in the PBS was registered, and its frequency in the sequenced DNA clones was calculated. The bar graph shows the frequencies of mutations for single-nucleotide (1nt) changes, double-nucleotide (2nt) changes, or changes involving three or more nucleotides (3nt).
    Figure Legend Snippet: Development of HIV-1 resistance to Cas9/sgPBS1. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS1-expressing SupT1 cells were harvested. Viral RNA was extracted and subjected to RT-PCR and sequencing. Each indel in the PBS was registered, and its frequency in the sequenced DNA clones was calculated. The bar graph shows the frequencies of mutations for single-nucleotide (1nt) changes, double-nucleotide (2nt) changes, or changes involving three or more nucleotides (3nt).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay

    71) Product Images from "HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site"

    Article Title: HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.01135-18

    Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.
    Figure Legend Snippet: Sequencing of tRNA 3 Lys genes in SupT1 cells that express Cas9 and sgPBS1, sgPBS2, or sgPBS3. Control cells express Cas9 only. (A) Illustration of tRNA 3 Lys binding to the HIV-1 PBS region (highlighted in green). (B) Incomplete complementation of sgPBS1, sgPBS2, or sgPBS3 to the tRNALys-TTT-3-5 gene. (C) PCR products of amplified tRNA 3 Lys genes from control SupT1 cells or SupT1 cells expressing Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. Sizes of DNA markers are shown on the left of the agarose gels. (D) Sanger sequencing data of the PCR products. The reference sequence for each tRNA 3 Lys gene was obtained from GenBank. (E) PCR products for gene tRNALys-TTT-3-5 in the control or Cas9/sgPBS-expressing SupT1 cells were cloned. About 24 DNA clones for each PCR product were sequenced. The frequency of each variant is presented.

    Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification, Expressing, Clone Assay, Variant Assay

    Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.
    Figure Legend Snippet: Escape of HIV-1 from inhibition by Cas9/sgPBS1, Cas9/sgPBS2, or Cas9/sgPBS3. (A) HIV-1 that was produced by transfecting HEK293T cells was used to infect SupT1 cells expressing Cas9 and sgPBS1, sgPBS2, or sgPBS3. Viral replication was monitored for a prolonged period of time by measuring viral RT activity in the culture supernatants at various time intervals. Control (ctrl) SupT1 cells expressed Cas9 only. This is called the first (1st) round of viral replication. (B) Viruses at the peak of the 1st round of viral replication in each cell line were collected and used to infect the same cell line using viruses with the same amounts of viral RT activity. This is called the second (2nd) round of viral replication. (C) The third (3rd) round of viral replication was performed using viruses collected at the peak of the second round to infect the same cell line. Results shown represent two independent replication experiments.

    Techniques Used: Inhibition, Produced, Expressing, Activity Assay

    The M3 and M4 PBS mutations resist inhibition by Cas9/sgPBS2. (A) Illustration of the M3 and M4 mutations in the PBS. Also shown are mutations G2557A (S3N in RT) and G4405A (G59E in integrase [IN]), which were detected in viruses that escaped from Cas9/sgPBS2. (B) The M3 and M4 mutants were refractory to Cas9/sgPBS2 in short-term infections. Wild-type and mutated HIV-1 were used to infect control or Cas9/sgPBS2-expressing SupT1 cells. Forty hours later, the levels of infectious HIV-1 in the culture supernatants were determined by infecting the TZM-bl cells. Results shown are the average values from three independent infections. Levels of each virus from the control cells are set at 100. *, P
    Figure Legend Snippet: The M3 and M4 PBS mutations resist inhibition by Cas9/sgPBS2. (A) Illustration of the M3 and M4 mutations in the PBS. Also shown are mutations G2557A (S3N in RT) and G4405A (G59E in integrase [IN]), which were detected in viruses that escaped from Cas9/sgPBS2. (B) The M3 and M4 mutants were refractory to Cas9/sgPBS2 in short-term infections. Wild-type and mutated HIV-1 were used to infect control or Cas9/sgPBS2-expressing SupT1 cells. Forty hours later, the levels of infectious HIV-1 in the culture supernatants were determined by infecting the TZM-bl cells. Results shown are the average values from three independent infections. Levels of each virus from the control cells are set at 100. *, P

    Techniques Used: Inhibition, Expressing

    Development of HIV-1 resistance to Cas9/sgPBS2. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS2-expressing SupT1 cells were collected and sequenced for the PBS DNA. The detected indels are shown, and their frequencies are presented. The bar graphs summarize the percentages of indels involving changes of one nucleotide (1nt), two nucleotides (2nt), or three or more nucleotides (3nt).
    Figure Legend Snippet: Development of HIV-1 resistance to Cas9/sgPBS2. Viruses at the peaks of the first, second, and third rounds of replication in the Cas9/sgPBS2-expressing SupT1 cells were collected and sequenced for the PBS DNA. The detected indels are shown, and their frequencies are presented. The bar graphs summarize the percentages of indels involving changes of one nucleotide (1nt), two nucleotides (2nt), or three or more nucleotides (3nt).

    Techniques Used: Expressing

    72) Product Images from "Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells"

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1152

    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P
    Figure Legend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    ( A ) Heatmap showing GRO-Seq, Pol2-S2P, γH2AX ChIP-Seq, and 8-oxodG OxiDIP-Seq signals at the gene body, and within the 5 kb both upstream the TSS and downstream the TTS of the RefSeq genes. Gene clusters identified by SeqMINER unbiased k-means clustering (Cluster #1–#4), and the number of genes contained within each cluster, as indicated. ( B ) Read density profiles of 8-oxodG OxiDIP-Seq (red) and γH2AX ChIP-Seq (black) in Cluster #1–#4, as indicated. ( C ) Box plot showing the distribution of transcription levels (RPKM, measured by GRO-seq) of human genes within each cluster in MCF10A cells. ( D ) Box plot showing the length distribution of human genes within each cluster in MCF10A cells ( P
    Figure Legend Snippet: ( A ) Heatmap showing GRO-Seq, Pol2-S2P, γH2AX ChIP-Seq, and 8-oxodG OxiDIP-Seq signals at the gene body, and within the 5 kb both upstream the TSS and downstream the TTS of the RefSeq genes. Gene clusters identified by SeqMINER unbiased k-means clustering (Cluster #1–#4), and the number of genes contained within each cluster, as indicated. ( B ) Read density profiles of 8-oxodG OxiDIP-Seq (red) and γH2AX ChIP-Seq (black) in Cluster #1–#4, as indicated. ( C ) Box plot showing the distribution of transcription levels (RPKM, measured by GRO-seq) of human genes within each cluster in MCF10A cells. ( D ) Box plot showing the length distribution of human genes within each cluster in MCF10A cells ( P

    Techniques Used: Chromatin Immunoprecipitation

    73) Product Images from "Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer"

    Article Title: Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-17-0593

    Analytical process and characteristics of the cohort of cases of acquired resistance to immune checkpoint inhibitors (A) Schematic representation of the repeat biopsy program and sample analysis. Tumor specimens (and corresponding PDXs when available) collected at the time of resistance to ICIs and before treatment with immune checkpoint inhibitors along with germline DNA were analyzed using whole exome sequencing. For select samples with sufficient material, RNA sequencing and quantitative immunofluorescence were also performed. (B) Pie-chart illustrating the types of therapies received by patients in this study. (C) Swimmer’s plot indicating time of response, resistance to ICIs, and length of time on therapy for individual patients.
    Figure Legend Snippet: Analytical process and characteristics of the cohort of cases of acquired resistance to immune checkpoint inhibitors (A) Schematic representation of the repeat biopsy program and sample analysis. Tumor specimens (and corresponding PDXs when available) collected at the time of resistance to ICIs and before treatment with immune checkpoint inhibitors along with germline DNA were analyzed using whole exome sequencing. For select samples with sufficient material, RNA sequencing and quantitative immunofluorescence were also performed. (B) Pie-chart illustrating the types of therapies received by patients in this study. (C) Swimmer’s plot indicating time of response, resistance to ICIs, and length of time on therapy for individual patients.

    Techniques Used: Sequencing, RNA Sequencing Assay, Immunofluorescence

    74) Product Images from "Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells—Brief Report"

    Article Title: Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells—Brief Report

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.118.311252

    Increased thymus weight in males with depletion of the AR (androgen receptor) in epithelial cells (E-ARKO [epithelial cell-specific AR knockout]). A , Gating strategy for sorting thymic epithelial cells (TECs). B , Assessment of AR knockout by measurement of exon 2 genomic DNA in control (K5-Cre + ) and E-ARKO mice, in enriched CD3 + T cells from thymus (control, n=10; and E-ARKO, n=10) and sorted TECs (control, n=4; and E-ARKO, n=4). * P
    Figure Legend Snippet: Increased thymus weight in males with depletion of the AR (androgen receptor) in epithelial cells (E-ARKO [epithelial cell-specific AR knockout]). A , Gating strategy for sorting thymic epithelial cells (TECs). B , Assessment of AR knockout by measurement of exon 2 genomic DNA in control (K5-Cre + ) and E-ARKO mice, in enriched CD3 + T cells from thymus (control, n=10; and E-ARKO, n=10) and sorted TECs (control, n=4; and E-ARKO, n=4). * P

    Techniques Used: Knock-Out, Mouse Assay

    75) Product Images from "Transcription factors Tp73, Cebpd, Pax6, and Spi1 rather than DNA methylation regulate chronic transcriptomics changes after experimental traumatic brain injury"

    Article Title: Transcription factors Tp73, Cebpd, Pax6, and Spi1 rather than DNA methylation regulate chronic transcriptomics changes after experimental traumatic brain injury

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-018-0519-z

    Enrichment scores of Gene Set Enrichment Analysis (GSEA) of DNA methylation in gene promoters in the ( a ) perilesional cortex ( b ) ipsilateral hippocampus, and ( c ) ipsilateral thalamus at 3 months after TBI. GSEA indicated significant negative enrichment in the perilesional cortex (FDR q-val. 0.046), but not in the ipsilateral hippocampus (FDR q-val. 0.079) or ipsilateral thalamus (FDR q-val. 0.828)
    Figure Legend Snippet: Enrichment scores of Gene Set Enrichment Analysis (GSEA) of DNA methylation in gene promoters in the ( a ) perilesional cortex ( b ) ipsilateral hippocampus, and ( c ) ipsilateral thalamus at 3 months after TBI. GSEA indicated significant negative enrichment in the perilesional cortex (FDR q-val. 0.046), but not in the ipsilateral hippocampus (FDR q-val. 0.079) or ipsilateral thalamus (FDR q-val. 0.828)

    Techniques Used: DNA Methylation Assay

    SignaLink transcription regulatory network (TRN) of Pax6 and its targets in the perilesional cortex. SignaLink network analysis revealed 300 targets, of which 36 were upregulated and 59 were downregulated in the perilesional cortex 3 months post-TBI. Color codes: blue circle, downregulation after TBI; yellow circle, upregulation after TBI; white circle, no change in gene expression after TBI; green line, transcriptional interaction with Pax6 ; black line, inter-target interaction
    Figure Legend Snippet: SignaLink transcription regulatory network (TRN) of Pax6 and its targets in the perilesional cortex. SignaLink network analysis revealed 300 targets, of which 36 were upregulated and 59 were downregulated in the perilesional cortex 3 months post-TBI. Color codes: blue circle, downregulation after TBI; yellow circle, upregulation after TBI; white circle, no change in gene expression after TBI; green line, transcriptional interaction with Pax6 ; black line, inter-target interaction

    Techniques Used: Expressing

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    TCID50 Assay:

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: In a TCID50 assay, each sample is scored negative or positive, so while the in situ method described for AAV is clearly compatible with extracting episomal DNA from cells, it was uncertain whether recovery of lentiviral vector target DNA would be quantitative. .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method).

    Dissection:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: Tissue samples for DNA analysis were obtained approximately 6 weeks after the onset of student dissection during which the cadavers are kept at room temperature. .. Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Cell Culture:

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care
    Article Snippet: To estimate the analytical sensitivity of quantitative real-time PCR (qPCR) and RPA-LF, DNA was extracted from 1 × 105 cultured promastigotes spiked in dog blood and then serially diluted to the equivalent of 0.01 parasites per reaction. .. DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor.

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis
    Article Snippet: Artificial "Mycoplasma free" samples treated with DNeasy Purification Kit Fourfold dilution series of human Hep2 DNA purified by Blood & Cell Culture DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) of initial concentration 8.000 copies/μl were prepared. .. Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure.

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species
    Article Snippet: Nematodes were subsequently disrupted in a Mini-BeadBeater-1 (Biospec Products, Inc., Bartlesville, OK) for 10 sec at 2,500 rpm, and total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer. .. PCR primer design for A. fragariae: Universal primers rDNA2 (5′-TTGATTACGTCCCTGCCCTTT-3′) described in and rDNA1.58S (5′-ACGAGCCGAGTGATCCACCG-3′) described in were used to amplify the partial sequence of the 18S rRNA gene, the full sequence of the ITS1 region and a partial sequence of the 5.8S rRNA gene from template DNA extracted from cultured A. fragariae .

    Generated:

    Article Title: Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization, et al. Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization
    Article Snippet: .. Overall, Qiagen's DNeasy Blood & Tissue Kit generated the highest DNA yield. ..

    other:

    Article Title: 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles
    Article Snippet: Alternatively, DNeasy Blood & Tissue Kit was used to purify DNA from the swab.

    Polymerase Chain Reaction:

    Article Title: Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma
    Article Snippet: .. PCR analysis Total DNA was prepared from normal-appearing liver and tumor samples using the DNeasy Blood and Tissue Kit according to the manufacturer's directions, including the optional addition of RNase (Qiagen, Valencia, CA). ..

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis
    Article Snippet: Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure. .. Samples were diluted twice, because of elution step with 50 μl of elute buffer, and therefore 4 μl instead of two of each sample were used in the LightCycler PCR run.

    Article Title: Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization, et al. Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization
    Article Snippet: A DNA extraction method should ideally retain high DNA yields while successfully removing PCR inhibitors. .. Overall, Qiagen's DNeasy Blood & Tissue Kit generated the highest DNA yield.

    Article Title: Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)
    Article Snippet: .. To prepare ascidians genomic DNA for PCR and LAMP, the dissected ascidians prepared above were incubated with 10 ml of PBS at room temperature for 30 min. After brief centrifugation, 1 ml of each supernatant was transferred to a new tube, centrifuged, and total DNA was extracted using a DNeasy tissue kit (Qiagen, Valencia, California, USA). .. One microliter of extracted DNA dissolved in 20 µl of doubly distilled water was used as the template for the PCR and LAMP assays.

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species
    Article Snippet: Nematodes were subsequently disrupted in a Mini-BeadBeater-1 (Biospec Products, Inc., Bartlesville, OK) for 10 sec at 2,500 rpm, and total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer. .. Nucleic acids were not quantified prior to use in PCR amplifications.

    DNA Extraction:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: .. Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.). .. For the FFPE procedure, extraction with xylene was omitted but incubation at 90 °C to reverse formalin crosslinking was performed.

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care
    Article Snippet: Paragraph title: Parasites and DNA extraction. ... DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor.

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method). ..

    Article Title: Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization, et al. Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization
    Article Snippet: A DNA extraction method should ideally retain high DNA yields while successfully removing PCR inhibitors. .. Overall, Qiagen's DNeasy Blood & Tissue Kit generated the highest DNA yield.

    Article Title: Methods to maximise recovery of environmental DNA from water samples
    Article Snippet: .. Four days after sample collection, DNA extraction was conducted on samples using either Qiagen’s DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany) or PowerWater DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA). .. DNA extraction with the DNeasy kit followed Renshaw et al.’s (2015) modification except we eluted the DNA in 200 μl buffer AE (Qiagen).

    Isolation:

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care
    Article Snippet: .. DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor. .. In brief, DNA was isolated from Whatman FTA paper as follows: two 6-mm disposable skin biopsy punches were used to cut the absorbed/dried samples and the filter disc was resuspended in 200 μL water and placed in a heat block at 96°C for 2 minutes.

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: Assay sensitivity is directly correlated to the number of transduction events (and therefore the number of transduced cells) that can be analyzed in each qPCR reaction and therefore, given a fixed qPCR reaction volume, to the concentration of the isolated DNA from transduced cells. .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method).

    Transferring:

    Article Title: Isolation and preservation of schistosome eggs and larvae in RNAlater® facilitates genetic profiling of individuals
    Article Snippet: Individual larval stages or eggs were then captured in 2 μl of the water using a Gilson pipette and ejected into the bottom of a 0.2 ml Eppendorf tube. .. In the laboratory gDNA from each individual was extracted using the DNeasy Tissue kit (Qiagen).

    Microscopy:

    Article Title: Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)
    Article Snippet: The collected ascidians were washed several times with 0.2 µm-filtered artificial seawater, tunics were dissected adjacent to the 2 siphons, chopped into small pieces (~0.2×0.2 cm) and placed in tubes containing 20 ml of sterilized artificial seawater, incubated at room temperature overnight, and directly examined under microscope the presence of flagellates as previously described [ ]. .. To prepare ascidians genomic DNA for PCR and LAMP, the dissected ascidians prepared above were incubated with 10 ml of PBS at room temperature for 30 min. After brief centrifugation, 1 ml of each supernatant was transferred to a new tube, centrifuged, and total DNA was extracted using a DNeasy tissue kit (Qiagen, Valencia, California, USA).

    Article Title: Isolation and preservation of schistosome eggs and larvae in RNAlater® facilitates genetic profiling of individuals
    Article Snippet: Each tube was checked for the presence of the individual larval stage by visualization under a binocular microscope and then 5 μl of RNAlater ® was added to preserve the sample and the tube was sealed (Fig. ). .. In the laboratory gDNA from each individual was extracted using the DNeasy Tissue kit (Qiagen).

    Purification:

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis
    Article Snippet: Paragraph title: Artificial "Mycoplasma free" samples treated with DNeasy Purification Kit ... Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure.

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit, which cuts the bacterial genome into approximately 50-kb fragments before separation by agarose gel electrophoresis. .. The results obtained with DNA purified from the vancomycin-resistant E. faecium control strain are shown in Fig. A, and those obtained with DNA purified from E. faecium clinical isolate 5 (lanes 7 and 8) and E. faecalis clinical isolate 2 (lanes 9 and 10) are shown in Fig. .

    Sequencing:

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species
    Article Snippet: Nematodes were subsequently disrupted in a Mini-BeadBeater-1 (Biospec Products, Inc., Bartlesville, OK) for 10 sec at 2,500 rpm, and total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer. .. PCR primer design for A. fragariae: Universal primers rDNA2 (5′-TTGATTACGTCCCTGCCCTTT-3′) described in and rDNA1.58S (5′-ACGAGCCGAGTGATCCACCG-3′) described in were used to amplify the partial sequence of the 18S rRNA gene, the full sequence of the ITS1 region and a partial sequence of the 5.8S rRNA gene from template DNA extracted from cultured A. fragariae .

    Staining:

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: The reduced SYTO 9 staining data suggested that exposure to copper could be affecting bacterial DNA. .. Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit, which cuts the bacterial genome into approximately 50-kb fragments before separation by agarose gel electrophoresis.

    Plasmid Preparation:

    Article Title: Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma
    Article Snippet: PCR analysis Total DNA was prepared from normal-appearing liver and tumor samples using the DNeasy Blood and Tissue Kit according to the manufacturer's directions, including the optional addition of RNase (Qiagen, Valencia, CA). .. To detect the luciferase-bearing plasmid pLiLucB in the genomic DNA, the forward primer 5′GACCGTGACCTACATCGTC and the reverse primer 5′-CATGTCTGCTCGAAGCGGC were used to amplify the luciferase gene.

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method). ..

    Selection:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: Paragraph title: Tissue selection and DNA isolation ... Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Agarose Gel Electrophoresis:

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit, which cuts the bacterial genome into approximately 50-kb fragments before separation by agarose gel electrophoresis. .. The results obtained with DNA purified from the vancomycin-resistant E. faecium control strain are shown in Fig. A, and those obtained with DNA purified from E. faecium clinical isolate 5 (lanes 7 and 8) and E. faecalis clinical isolate 2 (lanes 9 and 10) are shown in Fig. .

    In Situ:

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method). ..

    Size-exclusion Chromatography:

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species
    Article Snippet: .. Nematodes were subsequently disrupted in a Mini-BeadBeater-1 (Biospec Products, Inc., Bartlesville, OK) for 10 sec at 2,500 rpm, and total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer. ..

    Preserving:

    Article Title: Methods to maximise recovery of environmental DNA from water samples
    Article Snippet: Paragraph title: Experiment 1: Comparison of different DNA capture, preservation and extraction combinations ... Four days after sample collection, DNA extraction was conducted on samples using either Qiagen’s DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany) or PowerWater DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA).

    Concentration Assay:

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis
    Article Snippet: Artificial "Mycoplasma free" samples treated with DNeasy Purification Kit Fourfold dilution series of human Hep2 DNA purified by Blood & Cell Culture DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) of initial concentration 8.000 copies/μl were prepared. .. Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure.

    Article Title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates
    Article Snippet: .. To model the recovery of episomal vector DNA from target cells, plasmid DNA of known concentration was “spiked” into a known quantity of assay target cells and total DNA was extracted using either the DNeasy Blood & Tissue kit (Qiagen) or a modified version of the AAV protocol for direct DNA extraction (in situ method). ..

    Formalin-fixed Paraffin-Embedded:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: .. Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.). .. For the FFPE procedure, extraction with xylene was omitted but incubation at 90 °C to reverse formalin crosslinking was performed.

    DNA Purification:

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care
    Article Snippet: Leishmania donovani reference strain (MHOM/IN/80/DD8) and six other strains of this species preserved in our cryobank were thawed for subsequent DNA purification. .. DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor.

    Lysis:

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care
    Article Snippet: DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor. .. Then, 20 μL proteinase K solution and 200 μL lysis buffer were added to the samples and incubated at 56°C for 10 minutes.

    Article Title: Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species
    Article Snippet: The nematode pellet, composed of mixed stages of juveniles and adults, was transferred to a 2.0 ml conical tube filled halfway with 1-mm glass beads and 180 μl of lysis buffer ATL (Qiagen, Inc., Valencia, CA). .. Nematodes were subsequently disrupted in a Mini-BeadBeater-1 (Biospec Products, Inc., Bartlesville, OK) for 10 sec at 2,500 rpm, and total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer.

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    Qiagen dneasy blood tissue kit
    Agarose gel electrophoresis of <t>DNA</t> isolated from cadaver tissues using the <t>DNeasy</t> Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment
    Dneasy Blood Tissue Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment

    Journal: BMC Medical Genomics

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    doi: 10.1186/s12920-016-0223-4

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment

    Article Snippet: Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Techniques: Agarose Gel Electrophoresis, Isolation

    Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Journal: BMC Medical Genomics

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    doi: 10.1186/s12920-016-0223-4

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Article Snippet: Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Techniques: Agarose Gel Electrophoresis, Isolation, Formalin-fixed Paraffin-Embedded

    Sensitivity of recombinase polymerase amplification–lateral flow (RPA-LF) to detect Leishmania infantum compared with real-time polymerase chain reaction (PCR) used as gold standard. Tenfold serial dilutions of L. infantum promastigotes in dog blood were extracted using Qiagen ® DNeasy blood and tissue kit and detected by real-time quantitative PCR (SYBRgreen) or RPA-LF. Parasite dilutions: 1 = 10 5 , 2 = 10 4 , 3 = 10 3 , 4 = 10 2 , 5 = 10, 6 = 1, and 7 = 0.1 parasites and Bl = uninfected dog blood. The top band is the control band; the lower band is the test band. This is a representative figure of two similar assays.

    Journal: The American Journal of Tropical Medicine and Hygiene

    Article Title: A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care

    doi: 10.4269/ajtmh.15-0145

    Figure Lengend Snippet: Sensitivity of recombinase polymerase amplification–lateral flow (RPA-LF) to detect Leishmania infantum compared with real-time polymerase chain reaction (PCR) used as gold standard. Tenfold serial dilutions of L. infantum promastigotes in dog blood were extracted using Qiagen ® DNeasy blood and tissue kit and detected by real-time quantitative PCR (SYBRgreen) or RPA-LF. Parasite dilutions: 1 = 10 5 , 2 = 10 4 , 3 = 10 3 , 4 = 10 2 , 5 = 10, 6 = 1, and 7 = 0.1 parasites and Bl = uninfected dog blood. The top band is the control band; the lower band is the test band. This is a representative figure of two similar assays.

    Article Snippet: DNA was isolated from blood or tissue samples using the QIAGEN DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA) following the instructions of the vendor.

    Techniques: Recombinase Polymerase Amplification, Flow Cytometry, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.

    Journal: BMC Microbiology

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis

    doi: 10.1186/1471-2180-4-35

    Figure Lengend Snippet: DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.

    Article Snippet: Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure.

    Techniques: Polymerase Chain Reaction

    Detection limit of Azumiobodo hoyamushi 18S rDNA LAMP assays (A). LAMP assays were performed using serial dilutions of A. hoyamushi genomic DNA (1 ng to 1 fg per reaction). Distilled water was used as a negative control. LAMP products were visualized by gel electrophoresis (B) and using Loopamp® fluorescent detection reagent (FD) (C). (B, C) Lane M, 100-bp DNA marker; lane 1, 1 ng; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg of A. hoyamushi genomic DNA; lane 8, distilled water; and lane 9, LAMP product after Mbo I digestion. (D-E) A. hoyamushi at a density of 1×10 3 parasites/µl was serially diluted and tested (D) using the LAMP assay (D) and by PCR (E) using F3 and B3 primers. Lane M, 100-bp DNA marker; lane 1, 1,000; lane 2, 100; lane 3, 10; lane 4, 1; lane 5, 0.1; lane 6, 0.01 of parasites per reaction; lane 7, distilled water. A. hoyamushi genomic DNA was prepared using DNeasy tissue kits (Qiagen) from in vitro cultured A. hoyamushi species [ 9 ] which were kindly provided by Dr. Kyung Il Park (Kunsan National University, Gunsan, Korea).

    Journal: The Korean Journal of Parasitology

    Article Title: Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

    doi: 10.3347/kjp.2014.52.3.305

    Figure Lengend Snippet: Detection limit of Azumiobodo hoyamushi 18S rDNA LAMP assays (A). LAMP assays were performed using serial dilutions of A. hoyamushi genomic DNA (1 ng to 1 fg per reaction). Distilled water was used as a negative control. LAMP products were visualized by gel electrophoresis (B) and using Loopamp® fluorescent detection reagent (FD) (C). (B, C) Lane M, 100-bp DNA marker; lane 1, 1 ng; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg of A. hoyamushi genomic DNA; lane 8, distilled water; and lane 9, LAMP product after Mbo I digestion. (D-E) A. hoyamushi at a density of 1×10 3 parasites/µl was serially diluted and tested (D) using the LAMP assay (D) and by PCR (E) using F3 and B3 primers. Lane M, 100-bp DNA marker; lane 1, 1,000; lane 2, 100; lane 3, 10; lane 4, 1; lane 5, 0.1; lane 6, 0.01 of parasites per reaction; lane 7, distilled water. A. hoyamushi genomic DNA was prepared using DNeasy tissue kits (Qiagen) from in vitro cultured A. hoyamushi species [ 9 ] which were kindly provided by Dr. Kyung Il Park (Kunsan National University, Gunsan, Korea).

    Article Snippet: To prepare ascidians genomic DNA for PCR and LAMP, the dissected ascidians prepared above were incubated with 10 ml of PBS at room temperature for 30 min. After brief centrifugation, 1 ml of each supernatant was transferred to a new tube, centrifuged, and total DNA was extracted using a DNeasy tissue kit (Qiagen, Valencia, California, USA).

    Techniques: Negative Control, Nucleic Acid Electrophoresis, Marker, Lamp Assay, Polymerase Chain Reaction, In Vitro, Cell Culture