gfp negative facs  (Qiagen)

 
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    Genomic DNA Buffer Set
    Description:
    For isolation of up to 100 µg high molecular weight DNA from blood and cultured cells Kit contents Qiagen Genomic DNA Buffer Set Genomic Tip Format Anion exchange Technology Manual Gravity Flow Processing Isolation of DNA up to 150 kb in Size No Phenol or Chloroform Extractions For 75 Mini 25 Midi or 10 Maxipreps using Qiagen Genomic tips Includes Buffers Including Specific Lysis Buffers for Yeast Bacteria Cells Blood and Tissue Y1 B1 B2 C1 G2 QBT QC QF Benefits Reliable isolation of DNA up to 150 kb in size No phenol or chloroform extractions Convenient parallel processing of multiple samples
    Catalog Number:
    19060
    Price:
    166
    Category:
    Blood Cell Culture DNA Midi Kit
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    Structured Review

    Qiagen gfp negative facs
    Genomic DNA Buffer Set
    For isolation of up to 100 µg high molecular weight DNA from blood and cultured cells Kit contents Qiagen Genomic DNA Buffer Set Genomic Tip Format Anion exchange Technology Manual Gravity Flow Processing Isolation of DNA up to 150 kb in Size No Phenol or Chloroform Extractions For 75 Mini 25 Midi or 10 Maxipreps using Qiagen Genomic tips Includes Buffers Including Specific Lysis Buffers for Yeast Bacteria Cells Blood and Tissue Y1 B1 B2 C1 G2 QBT QC QF Benefits Reliable isolation of DNA up to 150 kb in size No phenol or chloroform extractions Convenient parallel processing of multiple samples
    https://www.bioz.com/result/gfp negative facs/product/Qiagen
    Average 92 stars, based on 21639 article reviews
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    gfp negative facs - by Bioz Stars, 2020-07
    92/100 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 "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

    11) 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

    12) 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

    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 "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

    15) 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

    16) 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

    17) 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

    18) Product Images from "Expression of PD-1 by T cells in malignant glioma patients reflects exhaustion and activation"

    Article Title: Expression of PD-1 by T cells in malignant glioma patients reflects exhaustion and activation

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-1176

    CD3 + PD-1 + T cells from pooled glioma patient PBMCs produce higher levels of IFN-γ compared to the CD3 + PD-1 − T cell population after T cell activation. A) The amount of IFN- γ produced in CD3 + PD-1 − , and CD3 + PD-1 + T cells from four glioma patient PBMCs 24 hours after CD3/CD28 activation. The dotted bar represents CD3 + PD-1 − T cells that were not activated. The checkerboard bar represents CD3 + PD-1 − T cells that were activated. The horizontal stripes bar represents CD3 + PD-1 + T cells that were activated. The results presented are a representative experiment that has been repeated multiple times with similar findings. B) IFN- γ produced at 48hrs from the same set of cells as (A).
    Figure Legend Snippet: CD3 + PD-1 + T cells from pooled glioma patient PBMCs produce higher levels of IFN-γ compared to the CD3 + PD-1 − T cell population after T cell activation. A) The amount of IFN- γ produced in CD3 + PD-1 − , and CD3 + PD-1 + T cells from four glioma patient PBMCs 24 hours after CD3/CD28 activation. The dotted bar represents CD3 + PD-1 − T cells that were not activated. The checkerboard bar represents CD3 + PD-1 − T cells that were activated. The horizontal stripes bar represents CD3 + PD-1 + T cells that were activated. The results presented are a representative experiment that has been repeated multiple times with similar findings. B) IFN- γ produced at 48hrs from the same set of cells as (A).

    Techniques Used: Activation Assay, Produced

    PD-1 + T cells have decreased TCR diversity compared to PD-1 − cells A) The number of estimated unique TCR sequences in PD-1 − and PD-1 + populations in healthy donor PBMC, glioma patient PBMC, and glioma patient TILs. Each point represents a single patient. Circle points represent healthy donor PBMCs. Square points represent glioma patient PBMCs. Triangle points represent glioma patient TILs. (**P≤0.005). B) The frequency of a specific TCR rearrangement in the populations. The populations shown are the CD3 + PD-1 − or CD3 + PD-1 + populations from healthy donor PBMCs (dotted bar), glioma patient PBMCs (the checkerboard bar), and glioma patient TILs (slanted stripes bar)
    Figure Legend Snippet: PD-1 + T cells have decreased TCR diversity compared to PD-1 − cells A) The number of estimated unique TCR sequences in PD-1 − and PD-1 + populations in healthy donor PBMC, glioma patient PBMC, and glioma patient TILs. Each point represents a single patient. Circle points represent healthy donor PBMCs. Square points represent glioma patient PBMCs. Triangle points represent glioma patient TILs. (**P≤0.005). B) The frequency of a specific TCR rearrangement in the populations. The populations shown are the CD3 + PD-1 − or CD3 + PD-1 + populations from healthy donor PBMCs (dotted bar), glioma patient PBMCs (the checkerboard bar), and glioma patient TILs (slanted stripes bar)

    Techniques Used:

    High dimensional analysis of mass cytometry data shows elevated expression of markers of activation and exhaustion on CD3 + PD-1 + TILs and markers of activation and antigen experience on CD3 + PD-1 + PBMCs from malignant glioma patients A) A PCA plot showing CD3 + PD-1 + TILs (green dots) and PBMCs (red dots). Each dot represents an individual patient. B) tSNE plots showing which clusters the CD3 + PD-1 + TILs were grouped in (green box) and which clusters the CD3 + PD-1 + PBMCs were grouped into (red box) by the clustering algorithm. C) The median expression levels of various activation markers in TILs (green dots) and PBMCs (red dots). The same tSNE plot as (B) was colored to show which cells expressed the particular marker (*P≤0.05, **P≤0.005, ***P≤0.0005). D) Antigen Experience/Memory markers. E) Exhaustion markers. F) A heat map of the median expression pattern for each of the exhaustion, activation, and memory/antigen experience markers for each individual patient.
    Figure Legend Snippet: High dimensional analysis of mass cytometry data shows elevated expression of markers of activation and exhaustion on CD3 + PD-1 + TILs and markers of activation and antigen experience on CD3 + PD-1 + PBMCs from malignant glioma patients A) A PCA plot showing CD3 + PD-1 + TILs (green dots) and PBMCs (red dots). Each dot represents an individual patient. B) tSNE plots showing which clusters the CD3 + PD-1 + TILs were grouped in (green box) and which clusters the CD3 + PD-1 + PBMCs were grouped into (red box) by the clustering algorithm. C) The median expression levels of various activation markers in TILs (green dots) and PBMCs (red dots). The same tSNE plot as (B) was colored to show which cells expressed the particular marker (*P≤0.05, **P≤0.005, ***P≤0.0005). D) Antigen Experience/Memory markers. E) Exhaustion markers. F) A heat map of the median expression pattern for each of the exhaustion, activation, and memory/antigen experience markers for each individual patient.

    Techniques Used: Mass Cytometry, Expressing, Activation Assay, Marker

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) Product Images from "Astrocytes in Juvenile Neuronal Ceroid Lipofuscinosis (CLN3) display metabolic and calcium signaling abnormalities"

    Article Title: Astrocytes in Juvenile Neuronal Ceroid Lipofuscinosis (CLN3) display metabolic and calcium signaling abnormalities

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14545

    CLN3 mutation alters astrocyte Ca 2+ responses. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) for 24 h and loaded with the Ca 2+ indicator dye Fluo4-AM. Following a 5 min period for baseline readings, cells were exposed to 10mM glutamate (Glu) and the amplitude of the first Ca 2+ response was calculated. Results are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 9 biological replicates (*, p
    Figure Legend Snippet: CLN3 mutation alters astrocyte Ca 2+ responses. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) for 24 h and loaded with the Ca 2+ indicator dye Fluo4-AM. Following a 5 min period for baseline readings, cells were exposed to 10mM glutamate (Glu) and the amplitude of the first Ca 2+ response was calculated. Results are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 9 biological replicates (*, p

    Techniques Used: Mutagenesis

    Glutamate uptake is not altered in Cln3 Δex7/8 astrocytes. Primary WT and Cln3 Δex7/8 astrocytes were treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h. Cells were then exposed to 1mM glutamate, whereupon supernatants were collected 30 min and 2 h later to evaluate residual extracellular glutamate concentrations. Results were normalized to the 1mM glutamate control and are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 3 biological replicates.
    Figure Legend Snippet: Glutamate uptake is not altered in Cln3 Δex7/8 astrocytes. Primary WT and Cln3 Δex7/8 astrocytes were treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h. Cells were then exposed to 1mM glutamate, whereupon supernatants were collected 30 min and 2 h later to evaluate residual extracellular glutamate concentrations. Results were normalized to the 1mM glutamate control and are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 3 biological replicates.

    Techniques Used:

    Ca 2+ signaling is disrupted in Cln3 Δex7/8 neurons. Primary WT and Cln3 Δex7/8 neurons were loaded with the Ca 2+ indicator dye Fluo4-AM. Following a 5 min period for baseline recordings, neurons were treated with 25nM L-glutamic acid and evaluated for a 420 sec period. Measurements were obtained using AxioVision software with fluorescent intensity normalized to baseline values. Results are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 3 biological replicates (*, p
    Figure Legend Snippet: Ca 2+ signaling is disrupted in Cln3 Δex7/8 neurons. Primary WT and Cln3 Δex7/8 neurons were loaded with the Ca 2+ indicator dye Fluo4-AM. Following a 5 min period for baseline recordings, neurons were treated with 25nM L-glutamic acid and evaluated for a 420 sec period. Measurements were obtained using AxioVision software with fluorescent intensity normalized to baseline values. Results are presented as the mean ± standard error of the mean (SEM) combined from three independent experiments with a total of 3 biological replicates (*, p

    Techniques Used: Software

    CLN3 mutation does not affect astrocyte glycolytic activity. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h, whereupon glycolytic activity was examined using Seahorse Bioscience assays. (A) Glycolysis, (B) Maximum capacity, and (C) Glycolytic reserve was determined based on extracellular acidification rate (ECAR). Results are representative of three independent experiments with a total of 3 biological replicates (mean ± standard error of the mean (SEM).
    Figure Legend Snippet: CLN3 mutation does not affect astrocyte glycolytic activity. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h, whereupon glycolytic activity was examined using Seahorse Bioscience assays. (A) Glycolysis, (B) Maximum capacity, and (C) Glycolytic reserve was determined based on extracellular acidification rate (ECAR). Results are representative of three independent experiments with a total of 3 biological replicates (mean ± standard error of the mean (SEM).

    Techniques Used: Mutagenesis, Activity Assay

    Mitochondrial respiration is impaired in Cln3 Δex7/8 astrocytes. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h, whereupon oxidative phosphorylation (Ox Phos) activity was examined using Seahorse Bioscience assays. (A) Basal mitochondrial respiration and (B) ATP production was determined based on oxygen consumption rate (OCR). Results are representative of three independent experiments with a total of 3 biological replicates (mean ± standard error of the mean (SEM). (C) Mitochondrial biomass was determined by quantiating NADH dehydrogenase 3 (ND3) and cytochrome c oxidase subunit-1 (Cox-1) expressed as a ratio to genomic DNA. Results are presented as the mean ± SEM of 4 biological replicates. Significant differences between WT and Cln3 Δex7/8 astrocytes are denoted by asterisks (*, p
    Figure Legend Snippet: Mitochondrial respiration is impaired in Cln3 Δex7/8 astrocytes. Primary WT and Cln3 Δex7/8 astrocytes were unstimulated or treated with TNF-α and IL-1β (10ng/mL each) or C6 ceramide (5μM) and neuronal lysate (NL) for 24 h, whereupon oxidative phosphorylation (Ox Phos) activity was examined using Seahorse Bioscience assays. (A) Basal mitochondrial respiration and (B) ATP production was determined based on oxygen consumption rate (OCR). Results are representative of three independent experiments with a total of 3 biological replicates (mean ± standard error of the mean (SEM). (C) Mitochondrial biomass was determined by quantiating NADH dehydrogenase 3 (ND3) and cytochrome c oxidase subunit-1 (Cox-1) expressed as a ratio to genomic DNA. Results are presented as the mean ± SEM of 4 biological replicates. Significant differences between WT and Cln3 Δex7/8 astrocytes are denoted by asterisks (*, p

    Techniques Used: Activity Assay

    Proposed mechanism for aberrant astrocyte-neuron crosstalk during CLN3 disease. Glutamate uptake by astrocytes is an energy-demanding process and ATP production was significantly decreased in Cln3 Δex7/8 astrocytes, which coincided with impaired Ca 2+ signaling. Cln3 Δex7/8 neurons were hyper-sensitive to physiological concentrations of glutamate, which elicited heightened and prolonged Ca 2+ signals that with time may contribute to neuronal dysfunction and/or death. Gln, glutamine; Glu, glutamate; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter 1.
    Figure Legend Snippet: Proposed mechanism for aberrant astrocyte-neuron crosstalk during CLN3 disease. Glutamate uptake by astrocytes is an energy-demanding process and ATP production was significantly decreased in Cln3 Δex7/8 astrocytes, which coincided with impaired Ca 2+ signaling. Cln3 Δex7/8 neurons were hyper-sensitive to physiological concentrations of glutamate, which elicited heightened and prolonged Ca 2+ signals that with time may contribute to neuronal dysfunction and/or death. Gln, glutamine; Glu, glutamate; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter 1.

    Techniques Used:

    26) Product Images from "Exploiting collateral sensitivity controls growth of mixed culture of sensitive and resistant cells and decreases selection for resistant cells"

    Article Title: Exploiting collateral sensitivity controls growth of mixed culture of sensitive and resistant cells and decreases selection for resistant cells

    Journal: bioRxiv

    doi: 10.1101/2020.05.07.082073

    Comparing the transcriptional program of CAMA-1 and CAMA-1_ribociclib_resistant cells reveals collateral sensitivity to Wee-1 inhibition Panel A : Schematic representation of significantly altered Hallmark pathways in between untreated CAMA-1 and CAMA-1_ribociclib_resistant cell lines. Positive normalized enrichment scores (NES) corresponds to enriched pathways in CAMA-1 (blue circles), while pathways with negative NES values (red circles) are enriched in CAMA-1_ribociclib_resistant cells. Additional enrichment scores for Hallmark pathways can be found in Supplementary Table 5. Panel B : Heatmap of all genes included in the Hallmark G2/M pathway in untreated CAMA-1 and CAMA-1_ribociclib_resistant cells. Representative genes of the pathway are labeled. Heatmap with all genes labeled can be found in Supplementary Figure 2. Panel C : Dose-response curves of CAMA-1 and CAMA-1_ribociclib_resistant cells under different concentrations of Wee-1 inhibitor adavosertib treatment. Cells were treated with increasing concentration of adavosertib for 96 hours, after which viability was measured using CellTiterGlo Chemiluminescent kit. The measured luminescence was normalized to the average of the lowest applied concentration (0.01 nM). Data points show the average of three replicates, error bars show standard deviation if it is larger than the size of the data point.
    Figure Legend Snippet: Comparing the transcriptional program of CAMA-1 and CAMA-1_ribociclib_resistant cells reveals collateral sensitivity to Wee-1 inhibition Panel A : Schematic representation of significantly altered Hallmark pathways in between untreated CAMA-1 and CAMA-1_ribociclib_resistant cell lines. Positive normalized enrichment scores (NES) corresponds to enriched pathways in CAMA-1 (blue circles), while pathways with negative NES values (red circles) are enriched in CAMA-1_ribociclib_resistant cells. Additional enrichment scores for Hallmark pathways can be found in Supplementary Table 5. Panel B : Heatmap of all genes included in the Hallmark G2/M pathway in untreated CAMA-1 and CAMA-1_ribociclib_resistant cells. Representative genes of the pathway are labeled. Heatmap with all genes labeled can be found in Supplementary Figure 2. Panel C : Dose-response curves of CAMA-1 and CAMA-1_ribociclib_resistant cells under different concentrations of Wee-1 inhibitor adavosertib treatment. Cells were treated with increasing concentration of adavosertib for 96 hours, after which viability was measured using CellTiterGlo Chemiluminescent kit. The measured luminescence was normalized to the average of the lowest applied concentration (0.01 nM). Data points show the average of three replicates, error bars show standard deviation if it is larger than the size of the data point.

    Techniques Used: Inhibition, Labeling, Concentration Assay, Standard Deviation

    Transcriptional response to ribociclib in sensitive and ribociclib-resistant cells Panel A : Dose-response curves of CAMA-1 and CAMA-1_ribociclib_resistant cells under different concentrations of ribociclib treatment. Cells were treated with increasing concentration of ribociclib for 96 hours, after which viability was measured using CellTiterGlo Chemiluminescent kit. The measured luminescence was normalized to the average of the lowest applied concentration (0.01 nM). Data points show the average of three replicates, error bars show standard deviation if it is larger than the size of the data point. Panel B : Venn diagram demonstrating the number of significantly differentially expressed genes in response to 12 hours of 1 µM ribociclib treatment in CAMA-1 and CAMA-1_ribociclib_resistant cells. Blue circle incorporates differentially expressed genes in CAMA-1, while the red circle incorporates differentially expressed genes in CAMA-1_ribociclib_resistant cells. Red numbers demonstrate the number of upregulated, while blue numbers demonstrate the number of downregulated genes in response to ribociclib treatment. Panel C and D : Heatmaps demonstrating the expression of significantly differentially expressed genes in response to ribociclib treatment in CAMA-1_ribociclib_resistant (Panel C) and CAMA-1 (Panel D) cells.
    Figure Legend Snippet: Transcriptional response to ribociclib in sensitive and ribociclib-resistant cells Panel A : Dose-response curves of CAMA-1 and CAMA-1_ribociclib_resistant cells under different concentrations of ribociclib treatment. Cells were treated with increasing concentration of ribociclib for 96 hours, after which viability was measured using CellTiterGlo Chemiluminescent kit. The measured luminescence was normalized to the average of the lowest applied concentration (0.01 nM). Data points show the average of three replicates, error bars show standard deviation if it is larger than the size of the data point. Panel B : Venn diagram demonstrating the number of significantly differentially expressed genes in response to 12 hours of 1 µM ribociclib treatment in CAMA-1 and CAMA-1_ribociclib_resistant cells. Blue circle incorporates differentially expressed genes in CAMA-1, while the red circle incorporates differentially expressed genes in CAMA-1_ribociclib_resistant cells. Red numbers demonstrate the number of upregulated, while blue numbers demonstrate the number of downregulated genes in response to ribociclib treatment. Panel C and D : Heatmaps demonstrating the expression of significantly differentially expressed genes in response to ribociclib treatment in CAMA-1_ribociclib_resistant (Panel C) and CAMA-1 (Panel D) cells.

    Techniques Used: Concentration Assay, Standard Deviation, Expressing

    27) Product Images from "Comparison of DNA extraction methods for non-marine molluscs: Is modified CTAB DNA extraction method more efficient than DNA extraction kits?"

    Article Title: Comparison of DNA extraction methods for non-marine molluscs: Is modified CTAB DNA extraction method more efficient than DNA extraction kits?

    Journal: bioRxiv

    doi: 10.1101/863167

    Comparison of average sequence quality between CTAB and Qiagen® DNeasy Blood and Tissue Kit extracted amplified genomic DNA
    Figure Legend Snippet: Comparison of average sequence quality between CTAB and Qiagen® DNeasy Blood and Tissue Kit extracted amplified genomic DNA

    Techniques Used: Sequencing, Amplification

    Chromatogram depicting 16S gene sequence quality in Qiagen ® DNeasy Blood and Tissue Kit extracted DNA.
    Figure Legend Snippet: Chromatogram depicting 16S gene sequence quality in Qiagen ® DNeasy Blood and Tissue Kit extracted DNA.

    Techniques Used: Sequencing

    28) Product Images from "Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens"

    Article Title: Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

    Journal: bioRxiv

    doi: 10.1101/871970

    Complementary CRISPR knockout and activation screens identify determinants of PARPi response in parental or BRCA2-knockout HeLa cells. (A) Schematic representation of the CRISPR knockout screen for olaparib sensitivity in wildtype cells. HeLa cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. Genomic DNA was extracted from cells surviving the drug treatment and single-guide RNAs (sgRNAs) were identified using Illumina sequencing. (B) Scatterplot showing the results of this screen. Each gene targeted by the library was ranked according to P-values calculated using RSA analysis. The P-values are based on the fold change of the guides targeting each gene between the olaparib- and DMSO-treated conditions. Several biologically interesting hits are highlighted. (C) Schematic representation of the CRISPR knockout screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. (D) Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. (E) Schematic representation of the CRISPR activation screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells stably expressing the modified dCas9 enzyme were infected with the Calabrese CRISPR activation library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. (F) Scatterplot showing the results of this screen, with several biologically interesting hits highlighted.
    Figure Legend Snippet: Complementary CRISPR knockout and activation screens identify determinants of PARPi response in parental or BRCA2-knockout HeLa cells. (A) Schematic representation of the CRISPR knockout screen for olaparib sensitivity in wildtype cells. HeLa cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. Genomic DNA was extracted from cells surviving the drug treatment and single-guide RNAs (sgRNAs) were identified using Illumina sequencing. (B) Scatterplot showing the results of this screen. Each gene targeted by the library was ranked according to P-values calculated using RSA analysis. The P-values are based on the fold change of the guides targeting each gene between the olaparib- and DMSO-treated conditions. Several biologically interesting hits are highlighted. (C) Schematic representation of the CRISPR knockout screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. (D) Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. (E) Schematic representation of the CRISPR activation screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells stably expressing the modified dCas9 enzyme were infected with the Calabrese CRISPR activation library. Infected cells were divided into PARP inhibitor (olaparib) -treated or control (DMSO) arms. (F) Scatterplot showing the results of this screen, with several biologically interesting hits highlighted.

    Techniques Used: CRISPR, Knock-Out, Activation Assay, Infection, Sequencing, Stable Transfection, Expressing, Modification

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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:

    38) Product Images from "Efficient Nonviral Stable Transgenesis Mediated by Retroviral Integrase"

    Article Title: Efficient Nonviral Stable Transgenesis Mediated by Retroviral Integrase

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.04.020

    Stable Transgenesis Using pLTR Vector in Culture Cells (A) A549 and PANC-1 cells were transfected with transgene cassette of pLTR-CMV:EGFP-pA and either pCS2-integrase-2A-tdTomato (indicated as integrase) or pCS2-tdTomato (indicated as control) expression plasmid. The images were acquired at three passages after transfection, and the expression of green fluorescence showed successful stable integration. (B) Normalized percentage of positive cells was analyzed by imaging process using ImageJ. ∗p
    Figure Legend Snippet: Stable Transgenesis Using pLTR Vector in Culture Cells (A) A549 and PANC-1 cells were transfected with transgene cassette of pLTR-CMV:EGFP-pA and either pCS2-integrase-2A-tdTomato (indicated as integrase) or pCS2-tdTomato (indicated as control) expression plasmid. The images were acquired at three passages after transfection, and the expression of green fluorescence showed successful stable integration. (B) Normalized percentage of positive cells was analyzed by imaging process using ImageJ. ∗p

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Fluorescence, Imaging

    39) 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

    40) 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

    Related Articles

    DNA Extraction:

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    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Amplification:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Mutagenesis:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Isolation:

    Article Title: In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice
    Article Snippet: .. Generation of the BbIVET library Total genomic DNA was isolated from a 250 ml culture of B. burgdorferi B31 clone A3 grown to a density 1×108 spirochetes/ml using the Qiagen genomic DNA buffer set and Genomic-tip 500/G, according to the manufacturer's protocol (Qiagen). .. A3 genomic DNA was partially digested with Tsp509I (New England Biolabs).

    Article Title: Identifying Phase-specific Genes in the Fungal Pathogen Histoplasma capsulatum Using a Genomic Shotgun Microarray D⃞
    Article Snippet: .. Genomic DNA was isolated from 100 ml of the virulent G217B strain by using genomic tips and genomic DNA buffer set (both from QIAGEN, Valencia, CA). .. For construction of the mini-array, DNA was partially digested with Sau3A I and size fractionated (0.5–2 kb) on a 1% low melt agarose gel.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Purification:

    Article Title: Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway
    Article Snippet: .. Genomic DNA of RS1, RS2, and their parental strain, MC4100, were purified with a Qiagen Genomic-tip 100/G and a Genomic DNA Buffer Set (Qiagen Inc., Valencia, CA), using the manufacturer's protocols. .. To remove high-affinity DNA-binding proteins and to ensure better DNA quality for the whole-genome sequence, the DNA solution was purified in two sequential steps: (i) DNA was first treated with a mixture of equal volumes of phenol and chloroform-isopropanol (24:1) to separate it into phenol and aqueous phases; (ii) the aqueous phase was mixed with an equal volume of chloroform-100% isopropanol (24:1 mixture).

    Polymerase Chain Reaction:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Sequencing:

    Article Title: Genome characterization of bile-isolated Shewanella algae ACCC
    Article Snippet: .. Library preparation, whole-genome sequence archive, and de novo assembly The bacterial genomic DNA was extracted from overnight culture of the S. algae ACCC using the QIAGEN Genomic-tip 100/G kit and Genomic DNA Buffer Set (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. .. Qubit dsDNA HS Assay kit and Qubit 2.0 fluorometer (Life Technologies) were used to measure DNA concentration.

    Plasmid Preparation:

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. DNA extraction, plasmid construction and PCR mutagenesis Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain
    Article Snippet: .. Ureaplasma genomic DNA was prepared by Genomic DNA Buffer Set (Qiagen, Hilden, Germany) and isolated by QIAGEN Genomic-tip 20/G (Qiagen, Hilden, Germany), which was then amplified by polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) for plasmid construction. .. To prepare the PCR product for the pUPV-230 plasmid construction, the primer pair UP162-F15 (5ʹ-ctagctagcatgatggacaattatgttag-3ʹ) and UP162-R15 (5ʹ-ccgctcgagatggcgtccattataaatatc-3ʹ) was used.

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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
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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