i pulchra  (Qiagen)

 
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    Name:
    QIAamp DNA Micro Kit
    Description:
    For purification of genomic and mitochondrial DNA from small samples Kit contents Qiagen QIAamp DNA Micro Kit 50 preps 1 to 100L Sample 20 to 100L Elution Volume Whole Blood Sample Silica Technology Manual Processing Spin Column Format Genomic DNA Mitochondrial DNA Purification 30 min Time Run 3g Yield Ideal for Real time PCR STR Analysis LMD PCR Analysis For Purification of Genomic and Mitochondrial DNA from Small Samples Includes 50 QIAamp MinElute Columns Proteinase K Carrier RNA Buffers 2mL Collection Tubes Benefits Rapid purification of high quality DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors
    Catalog Number:
    56304
    Price:
    237
    Category:
    QIAamp DNA Micro Kit
    Buy from Supplier


    Structured Review

    Qiagen i pulchra
    QIAamp DNA Micro Kit
    For purification of genomic and mitochondrial DNA from small samples Kit contents Qiagen QIAamp DNA Micro Kit 50 preps 1 to 100L Sample 20 to 100L Elution Volume Whole Blood Sample Silica Technology Manual Processing Spin Column Format Genomic DNA Mitochondrial DNA Purification 30 min Time Run 3g Yield Ideal for Real time PCR STR Analysis LMD PCR Analysis For Purification of Genomic and Mitochondrial DNA from Small Samples Includes 50 QIAamp MinElute Columns Proteinase K Carrier RNA Buffers 2mL Collection Tubes Benefits Rapid purification of high quality DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors
    https://www.bioz.com/result/i pulchra/product/Qiagen
    Average 91 stars, based on 5555 article reviews
    Price from $9.99 to $1999.99
    i pulchra - by Bioz Stars, 2020-05
    91/100 stars

    Images

    1) Product Images from "The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae"

    Article Title: The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01608-4

    Predicted secondary structure of tRNAs from the mitochondrial genome sequences of and Isodiametra pulchra as predicted by MiTFi in Mitos.
    Figure Legend Snippet: Predicted secondary structure of tRNAs from the mitochondrial genome sequences of and Isodiametra pulchra as predicted by MiTFi in Mitos.

    Techniques Used:

    Bayesian (using PhyloBayes 53 ) and Maximum Likelihood (using RAxML 51 ) phylogenetic analysis of mitochondrial protein-coding genes from the Metazoa, including P. rubra , I. pulchra and A. ylvae with posterior probability and bootstrap support values, respectively, at relevant nodes. Analysis carried out on trimmed alignment. Topology of both trees is identical.
    Figure Legend Snippet: Bayesian (using PhyloBayes 53 ) and Maximum Likelihood (using RAxML 51 ) phylogenetic analysis of mitochondrial protein-coding genes from the Metazoa, including P. rubra , I. pulchra and A. ylvae with posterior probability and bootstrap support values, respectively, at relevant nodes. Analysis carried out on trimmed alignment. Topology of both trees is identical.

    Techniques Used:

    Overview of the initial transcriptome assembly fragments and PCR strategy for scaffolding the Isodiametra pulchra mitochondrial genome. 1.3 kb, 13 kb and 3.5 kb fragments aligned to a continuous 19 kb fragment, with the location of the duplicated sequence in the 13 kb and 3.5 kb fragments shown by blue dashed lines. The ‘start’ and ‘end’ regions of the 13 kb and 3.5 kb fragments are annotated by 5′ (start) and 3′ (end). The approximate location of cob and nad1 protein-coding sequence are shown for reference. Reliable PCR-amplicons are shown in orange; the green PCR fragment indicates successful joining of the 3′ end of the 3.5 kb fragment to the rrnL fragment, including the duplicated section. The 18,725 base-pair long sequence we resolve is indicated by the pink lines, from ‘start’ to ‘end’.
    Figure Legend Snippet: Overview of the initial transcriptome assembly fragments and PCR strategy for scaffolding the Isodiametra pulchra mitochondrial genome. 1.3 kb, 13 kb and 3.5 kb fragments aligned to a continuous 19 kb fragment, with the location of the duplicated sequence in the 13 kb and 3.5 kb fragments shown by blue dashed lines. The ‘start’ and ‘end’ regions of the 13 kb and 3.5 kb fragments are annotated by 5′ (start) and 3′ (end). The approximate location of cob and nad1 protein-coding sequence are shown for reference. Reliable PCR-amplicons are shown in orange; the green PCR fragment indicates successful joining of the 3′ end of the 3.5 kb fragment to the rrnL fragment, including the duplicated section. The 18,725 base-pair long sequence we resolve is indicated by the pink lines, from ‘start’ to ‘end’.

    Techniques Used: Polymerase Chain Reaction, Scaffolding, Sequencing

    Overview of the mitochondrial genome sequences we resolve for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae (Xenacoelomorpha: Acoela). Genes not drawn to scale. Numbers beneath the sequences show intergenic spaces (positive values) or intergenic overlap (negative values). Protein-coding genes are denoted by three letter abbreviations; ribosomal genes by four letter abbreviations. tRNAs are shown by single uppercase letters. ( A ) P. rubra 14,957 base-pair long sequence. All genes found on the positive (forward) strand. Where genes, rRNAs or tRNAs are coloured orange, this is solely to demonstrate overlap with adjacent genes, rRNAs or tRNAs. ( B ) I. pulchra 18,725 base-pair long sequence. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding sequence shown in grey. ( C ) A. ylvae 16,619 nucleotide-long mitochondrial genome. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding regions greater than 100 nucleotides in length are shown in grey.
    Figure Legend Snippet: Overview of the mitochondrial genome sequences we resolve for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae (Xenacoelomorpha: Acoela). Genes not drawn to scale. Numbers beneath the sequences show intergenic spaces (positive values) or intergenic overlap (negative values). Protein-coding genes are denoted by three letter abbreviations; ribosomal genes by four letter abbreviations. tRNAs are shown by single uppercase letters. ( A ) P. rubra 14,957 base-pair long sequence. All genes found on the positive (forward) strand. Where genes, rRNAs or tRNAs are coloured orange, this is solely to demonstrate overlap with adjacent genes, rRNAs or tRNAs. ( B ) I. pulchra 18,725 base-pair long sequence. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding sequence shown in grey. ( C ) A. ylvae 16,619 nucleotide-long mitochondrial genome. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding regions greater than 100 nucleotides in length are shown in grey.

    Techniques Used: Sequencing

    Comparisons of gene orders in the mitochondrial genome sequences resolved for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae compared to a published P. rubra fragment; the acoel Symsagittifera roscoffensis ; the xenoturbellid Xenoturbella bocki ; the nemertodermatid Nemertoderma westbladi and the metazoan mitochondrial ‘ground plan’ gene order, represented by Limulus polyphemus . Genes are not drawn to scale. Coloured genes chosen to show ‘anchors’ and divergence from the ground plan gene order in other species.
    Figure Legend Snippet: Comparisons of gene orders in the mitochondrial genome sequences resolved for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae compared to a published P. rubra fragment; the acoel Symsagittifera roscoffensis ; the xenoturbellid Xenoturbella bocki ; the nemertodermatid Nemertoderma westbladi and the metazoan mitochondrial ‘ground plan’ gene order, represented by Limulus polyphemus . Genes are not drawn to scale. Coloured genes chosen to show ‘anchors’ and divergence from the ground plan gene order in other species.

    Techniques Used:

    2) Product Images from "Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation"

    Article Title: Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

    Journal: Learning & Memory

    doi: 10.1101/lm.047977.118

    Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,
    Figure Legend Snippet: Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,

    Techniques Used: Methylation

    3) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    4) Product Images from "Simultaneous Isolation of DNA and RNA from the Same Cell Population Obtained by Laser Capture Microdissection for Genome and Transcriptome Profiling"

    Article Title: Simultaneous Isolation of DNA and RNA from the Same Cell Population Obtained by Laser Capture Microdissection for Genome and Transcriptome Profiling

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.2353/jmoldx.2008.070131

    Using the DNA purified by the adapted AllPrep DNA/RNA method for genome profiling. A: Amplified tumor DNA fragments from DNA purified by the adapted AllPrep DNA/RNA method and by the QIAamp DNA purification method as revealed on a 2% agarose gel.
    Figure Legend Snippet: Using the DNA purified by the adapted AllPrep DNA/RNA method for genome profiling. A: Amplified tumor DNA fragments from DNA purified by the adapted AllPrep DNA/RNA method and by the QIAamp DNA purification method as revealed on a 2% agarose gel.

    Techniques Used: Purification, Amplification, DNA Purification, Agarose Gel Electrophoresis

    5) Product Images from "Central metabolism of functionally heterogeneous mesenchymal stromal cells"

    Article Title: Central metabolism of functionally heterogeneous mesenchymal stromal cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-51937-9

    Mitochondrial DNA copy number and methylation are molecular markers of LL- or SL-CBMSC identity. ( A ) LL-CBMSC showed high mtDNAcn compared to SL-CBMSC. Mean and standard deviation are represented; *p
    Figure Legend Snippet: Mitochondrial DNA copy number and methylation are molecular markers of LL- or SL-CBMSC identity. ( A ) LL-CBMSC showed high mtDNAcn compared to SL-CBMSC. Mean and standard deviation are represented; *p

    Techniques Used: Methylation, Standard Deviation

    SL-CBMSC as compared to LL-CBMSC show a decrease of mitochondrial OXPHOS expression and function associated to higher mitochondrial potential. Cells were cultured for 48 hours in standard growth condition and then analyzed. ( A ) LL-CBMSC showed higher mtDNAcn, assessed by qPCR. Mean and standard deviation are represented. ( B ) qPCR analysis of the expression levels of the indicated mRNA, codified by mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Actin was used as housekeeping gene to normalize the expression levels. ( C ) Western blot analysis of the expression levels of the indicated mitochondrial proteins. Vinculin was used to normalize the expression levels. Blots shown are derived from multiple gels run under the same experimental conditions. The membrane was cut based on molecular weight. All full length blots are presented in supplementary Fig. 4A . ( D , E ) Basal ( D ) and ATP production-coupled ( E ) oxygen consumption rate (OCR). The latter was calculated subtracting the OCR upon oligomycin to the basal OCR. Data are represented as mean ± SEM of the different cell populations. For each cell population n ≥ 2 biological analyses were performed with n ≥ 5 replicates for each. ( F ) Representative confocal microscopic images after staining with TMRE (50 nM). Scale bar 20 µm. ( G - H ) Quantification of TMRE signal using confocal microscopy ( G ) and flow cytometry ( H ). Mean and standard deviation are represented. For panels B-H, ns, not statistically significant; *p
    Figure Legend Snippet: SL-CBMSC as compared to LL-CBMSC show a decrease of mitochondrial OXPHOS expression and function associated to higher mitochondrial potential. Cells were cultured for 48 hours in standard growth condition and then analyzed. ( A ) LL-CBMSC showed higher mtDNAcn, assessed by qPCR. Mean and standard deviation are represented. ( B ) qPCR analysis of the expression levels of the indicated mRNA, codified by mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Actin was used as housekeeping gene to normalize the expression levels. ( C ) Western blot analysis of the expression levels of the indicated mitochondrial proteins. Vinculin was used to normalize the expression levels. Blots shown are derived from multiple gels run under the same experimental conditions. The membrane was cut based on molecular weight. All full length blots are presented in supplementary Fig. 4A . ( D , E ) Basal ( D ) and ATP production-coupled ( E ) oxygen consumption rate (OCR). The latter was calculated subtracting the OCR upon oligomycin to the basal OCR. Data are represented as mean ± SEM of the different cell populations. For each cell population n ≥ 2 biological analyses were performed with n ≥ 5 replicates for each. ( F ) Representative confocal microscopic images after staining with TMRE (50 nM). Scale bar 20 µm. ( G - H ) Quantification of TMRE signal using confocal microscopy ( G ) and flow cytometry ( H ). Mean and standard deviation are represented. For panels B-H, ns, not statistically significant; *p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Derivative Assay, Molecular Weight, Staining, Confocal Microscopy, Flow Cytometry, Cytometry

    6) Product Images from "PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5? Nuclease (TaqMan) Assay †"

    Article Title: PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5? Nuclease (TaqMan) Assay †

    Journal: Applied and Environmental Microbiology

    doi:

    Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER (□) and QIAamp tissue kit (▿) DNA extraction methods, and 5 μl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average ΔRQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The ΔRQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean ( n = 3).
    Figure Legend Snippet: Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER (□) and QIAamp tissue kit (▿) DNA extraction methods, and 5 μl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average ΔRQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The ΔRQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean ( n = 3).

    Techniques Used: Nuclease Assay, Incubation, DNA Extraction, Amplification, Sequencing, Standard Deviation

    7) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    8) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    9) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    10) Product Images from "A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides"

    Article Title: A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

    Journal: BMC Microbiology

    doi: 10.1186/s12866-018-1335-0

    a and b : Nested real-time PCR of the primary PCR amplicon amplified from pre-processing 30 catapulted cells from a glass slide using LCM. a Quantification cycles show early detection of undiluted primary amplicon compared to the positive control. The positive control was diluted at 10-fold to avoid primer saturation. b Agarose (1.5%, 4 °C) gel electrophoresis showing the 176 bp nested real-time PCR amplification. The amplification was performed by using primers BnMS949bf and 1105br. Lane 1 and 7: 100 bp ladder; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control
    Figure Legend Snippet: a and b : Nested real-time PCR of the primary PCR amplicon amplified from pre-processing 30 catapulted cells from a glass slide using LCM. a Quantification cycles show early detection of undiluted primary amplicon compared to the positive control. The positive control was diluted at 10-fold to avoid primer saturation. b Agarose (1.5%, 4 °C) gel electrophoresis showing the 176 bp nested real-time PCR amplification. The amplification was performed by using primers BnMS949bf and 1105br. Lane 1 and 7: 100 bp ladder; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Laser Capture Microdissection, Positive Control, Nucleic Acid Electrophoresis, Ethanol Precipitation, Negative Control

    (i, ii and iii): Agarose gel electrophoresis (1.5%, 4 °C) showing the amplification process of 30 M. smegmatis (NCIMB 8548) cells catapulted from Ziehl-Neelsen archived slide using LCM. (i) Multiple displacement amplification (MDA) by using REPLI-g® UltraFast Mini kit (Qiagen). Samples were extracted using 3 different methods to determine an appropriate method for a lower number of cells isolated from archived glass slides. Lane 1 and 6: 100 bp ladder; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control. (ii) 600 bp product of primary PCR using post-MDA mixture as a template which was performed by using touchdown PCR. Lane 1 and 7: 100 bp ladders; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control. (iii) 176 bp product of touchdown nested PCR amplified from primary amplicon. Lane 1 and 7: The 100 bp ladders; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control
    Figure Legend Snippet: (i, ii and iii): Agarose gel electrophoresis (1.5%, 4 °C) showing the amplification process of 30 M. smegmatis (NCIMB 8548) cells catapulted from Ziehl-Neelsen archived slide using LCM. (i) Multiple displacement amplification (MDA) by using REPLI-g® UltraFast Mini kit (Qiagen). Samples were extracted using 3 different methods to determine an appropriate method for a lower number of cells isolated from archived glass slides. Lane 1 and 6: 100 bp ladder; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control. (ii) 600 bp product of primary PCR using post-MDA mixture as a template which was performed by using touchdown PCR. Lane 1 and 7: 100 bp ladders; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control. (iii) 176 bp product of touchdown nested PCR amplified from primary amplicon. Lane 1 and 7: The 100 bp ladders; Lane 2: Heat-shock; Lane 3: Heat-shock followed by ethanol precipitation; Lane 4: QIAamp DNA Micro kit; Lane 5: positive control; Lane 6: negative control

    Techniques Used: Agarose Gel Electrophoresis, Amplification, Laser Capture Microdissection, Multiple Displacement Amplification, Isolation, Ethanol Precipitation, Positive Control, Polymerase Chain Reaction, Touchdown PCR, Negative Control, Nested PCR

    11) Product Images from "Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling"

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0367-z

    Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining, Isolation, Sequencing

    Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Expressing, Isolation, Transfection, Clone Assay, Marker, Confocal Microscopy, Staining

    Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Expressing, Electrophoresis, Staining, Marker, Confocal Microscopy, Mouse Assay

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

    12) Product Images from "Novel biomarker ZCCHC13 revealed by integrating DNA methylation and mRNA expression data in non-obstructive azoospermia"

    Article Title: Novel biomarker ZCCHC13 revealed by integrating DNA methylation and mRNA expression data in non-obstructive azoospermia

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0033-x

    Schematic pipeline depicting the strategy of identifying key genes from DNA methylation and mRNA expression microarray data. The method proceeds as follows:(1) identify significantly differentially methylated genes and expressed genes in the case and control groups; (2) retain those genes whose methylation and expression levels are highly anti-correlated; (3) enrich the gene ontology, KEGG pathway, and PPI networks of anti-correlated genes; (4) identify key genes from the intersection of statistical analysis, gene functional annotations, and interaction networks
    Figure Legend Snippet: Schematic pipeline depicting the strategy of identifying key genes from DNA methylation and mRNA expression microarray data. The method proceeds as follows:(1) identify significantly differentially methylated genes and expressed genes in the case and control groups; (2) retain those genes whose methylation and expression levels are highly anti-correlated; (3) enrich the gene ontology, KEGG pathway, and PPI networks of anti-correlated genes; (4) identify key genes from the intersection of statistical analysis, gene functional annotations, and interaction networks

    Techniques Used: DNA Methylation Assay, Expressing, Microarray, Methylation, Functional Assay

    13) Product Images from "A 22q11.2 amplification in the region encoding microRNA-650 correlates with the epithelial to mesenchymal transition in breast cancer primary cultures of Mexican patients"

    Article Title: A 22q11.2 amplification in the region encoding microRNA-650 correlates with the epithelial to mesenchymal transition in breast cancer primary cultures of Mexican patients

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2017.3842

    LOH and gain and losses present in breast cancer primary cultures of Mexican patients. (A) LOH alterations in individual and in common in two samples. There were no LOH alterations in common in the three samples analyzed. (B) Gains and losses alterations in individual, and common in two and three samples. Analysis performed with the microarray CytoScan HD and the software Chromosome Analysis Suite 3.0. The gain of the region 22q11.2 that encodes for miR-650 was present in all the samples analyzed.
    Figure Legend Snippet: LOH and gain and losses present in breast cancer primary cultures of Mexican patients. (A) LOH alterations in individual and in common in two samples. There were no LOH alterations in common in the three samples analyzed. (B) Gains and losses alterations in individual, and common in two and three samples. Analysis performed with the microarray CytoScan HD and the software Chromosome Analysis Suite 3.0. The gain of the region 22q11.2 that encodes for miR-650 was present in all the samples analyzed.

    Techniques Used: Microarray, Software

    14) Product Images from "Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells"

    Article Title: Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.3003761

    Transcriptional activity and CAR function in persisting cells (A) The CD4ζ RNA level (y axis) is plotted versus the number of DNA CD4ζ DNA copies per million PBMC of each tested sample. Samples from the Deeks, Mitsuyasu and Aronson studies are plotted as red, blue and green symbols, respectively. CD4ζ RNA expression was calculated from the ΔCt values for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is expressed at a high level, so that greater expression of CD4ζ results in a smaller expression difference and so a smaller ΔCt. The values are significantly correlated by linear regression analysis (p=0.0018) testing whether rho=0 or not. No RT-controls were run in parallel and all plotted CD4ζ samples were negative, confirming the signal observed is due to RNA template. Two subjects did not have detectable CD4ζ RNA. (B) . Functionality is measured as the relative increase in the average copy number of CD4ζ cells following anti-CD4 antibody activation over percentage of CD4ζ before stimulation. (C) Fold-increase of CD4ζ expressing cells following three 10-day rounds of anti-CD4 mAb loaded irradiated K562 artificial antigen presenting cells expressing the high affinity Fc Receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ copy numbers were evaluated from the gDNA of subject PBMCs before and after activation by qPCR analysis. The final percentage of CD4ζ in each culture is indicated by the number at the top of each bar. Each bar is designated at the bottom with the subject ID and year post-infusion of the sample.
    Figure Legend Snippet: Transcriptional activity and CAR function in persisting cells (A) The CD4ζ RNA level (y axis) is plotted versus the number of DNA CD4ζ DNA copies per million PBMC of each tested sample. Samples from the Deeks, Mitsuyasu and Aronson studies are plotted as red, blue and green symbols, respectively. CD4ζ RNA expression was calculated from the ΔCt values for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is expressed at a high level, so that greater expression of CD4ζ results in a smaller expression difference and so a smaller ΔCt. The values are significantly correlated by linear regression analysis (p=0.0018) testing whether rho=0 or not. No RT-controls were run in parallel and all plotted CD4ζ samples were negative, confirming the signal observed is due to RNA template. Two subjects did not have detectable CD4ζ RNA. (B) . Functionality is measured as the relative increase in the average copy number of CD4ζ cells following anti-CD4 antibody activation over percentage of CD4ζ before stimulation. (C) Fold-increase of CD4ζ expressing cells following three 10-day rounds of anti-CD4 mAb loaded irradiated K562 artificial antigen presenting cells expressing the high affinity Fc Receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ copy numbers were evaluated from the gDNA of subject PBMCs before and after activation by qPCR analysis. The final percentage of CD4ζ in each culture is indicated by the number at the top of each bar. Each bar is designated at the bottom with the subject ID and year post-infusion of the sample.

    Techniques Used: Activity Assay, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay, Irradiation, Real-time Polymerase Chain Reaction

    15) Product Images from "Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling"

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0367-z

    Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining, Isolation, Sequencing

    Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Expressing, Isolation, Transfection, Clone Assay, Marker, Confocal Microscopy, Staining

    Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Expressing, Electrophoresis, Staining, Marker, Confocal Microscopy, Mouse Assay

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

    16) Product Images from "Microbial Signatures and Innate Immune Gene Expression in Lamina Propria Phagocytes of Inflammatory Bowel Disease Patients"

    Article Title: Microbial Signatures and Innate Immune Gene Expression in Lamina Propria Phagocytes of Inflammatory Bowel Disease Patients

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2019.10.013

    Immune gene expression in CD11b+ cells relates to the inflammation state of the disease. ( A ) Principal component analysis (PCA) based on log 2 transformed gene expression counts obtained from inflamed and noninflamed lamina propria phagocytes of UC and CD patients (n = 6–14 samples per group). Numbers in parentheses indicate the percentage variation explained by the axis. ( B ) Venn diagram showing the number of differentially expressed genes that were shared between inflamed and noninflamed samples obtained from CD and UC patients. ( C and D ) Volcano plots showing genes differentially expressed between inflamed and noninflamed samples from CD and UC patients, respectively. Colored symbols in the volcano plots represent genes that were significantly more abundant (log 2 fold change greater than 1 and P adjusted ≤ .1) in the respective groups: CD inflamed (grey), CD noninflamed (green), UC inflamed (blue), UC noninflamed (pink). The P values were obtained by t test followed by adjustment for false-discovery rate. I, inflamed; NI, non-inflamed.
    Figure Legend Snippet: Immune gene expression in CD11b+ cells relates to the inflammation state of the disease. ( A ) Principal component analysis (PCA) based on log 2 transformed gene expression counts obtained from inflamed and noninflamed lamina propria phagocytes of UC and CD patients (n = 6–14 samples per group). Numbers in parentheses indicate the percentage variation explained by the axis. ( B ) Venn diagram showing the number of differentially expressed genes that were shared between inflamed and noninflamed samples obtained from CD and UC patients. ( C and D ) Volcano plots showing genes differentially expressed between inflamed and noninflamed samples from CD and UC patients, respectively. Colored symbols in the volcano plots represent genes that were significantly more abundant (log 2 fold change greater than 1 and P adjusted ≤ .1) in the respective groups: CD inflamed (grey), CD noninflamed (green), UC inflamed (blue), UC noninflamed (pink). The P values were obtained by t test followed by adjustment for false-discovery rate. I, inflamed; NI, non-inflamed.

    Techniques Used: Expressing, Transformation Assay

    Flow cytometric characterization of lamina propria CD11b+ cells isolated from biopsy specimens obtained from IBD patients. ( A ) The CD11b+ population was examined by determining the expression of CD33 (Siglec-3) (monocytes, macrophages, granulocytes, dendritic cells, and mast cells) and the activation marker CD206 (mannose receptor) (macrophages and dendritic cells). Cells also were assessed for expression of CD14 (part of the lipopolysaccharide-receptor complex) and HLA-DR (antigen presentation) in the 2 subsets. ( B ) Number (means ± 95% CI) of CD11b+ cells isolated from inflamed/uninflamed patient biopsy specimens. FSC-A, forward scatter area; SSC-A, side scatter area.
    Figure Legend Snippet: Flow cytometric characterization of lamina propria CD11b+ cells isolated from biopsy specimens obtained from IBD patients. ( A ) The CD11b+ population was examined by determining the expression of CD33 (Siglec-3) (monocytes, macrophages, granulocytes, dendritic cells, and mast cells) and the activation marker CD206 (mannose receptor) (macrophages and dendritic cells). Cells also were assessed for expression of CD14 (part of the lipopolysaccharide-receptor complex) and HLA-DR (antigen presentation) in the 2 subsets. ( B ) Number (means ± 95% CI) of CD11b+ cells isolated from inflamed/uninflamed patient biopsy specimens. FSC-A, forward scatter area; SSC-A, side scatter area.

    Techniques Used: Isolation, Expressing, Activation Assay, Marker

    17) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    18) Product Images from "Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection"

    Article Title: Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005153

    Detection of HIV DNA and RNA in adipose tissue. (A) Detection of HIV DNA in SVF cells and PBMCs from 11 ART-treated, HIV-infected patients. Each patient is represented by a symbol, and the shading represents the type of adipose tissue: SCAT (open symbols), VAT (filled symbols). (B) HIV DNA detection in sorted CD4 + T cells recovered from adipose tissue and PBMCs from 3 ART-treated, HIV-infected patients. The HIV DNA detection assay was performed in duplicate and is expressed in log copies per million cells. The detection limit differed as a function of the numbers of cells tested and is indicated as
    Figure Legend Snippet: Detection of HIV DNA and RNA in adipose tissue. (A) Detection of HIV DNA in SVF cells and PBMCs from 11 ART-treated, HIV-infected patients. Each patient is represented by a symbol, and the shading represents the type of adipose tissue: SCAT (open symbols), VAT (filled symbols). (B) HIV DNA detection in sorted CD4 + T cells recovered from adipose tissue and PBMCs from 3 ART-treated, HIV-infected patients. The HIV DNA detection assay was performed in duplicate and is expressed in log copies per million cells. The detection limit differed as a function of the numbers of cells tested and is indicated as

    Techniques Used: Infection, Detection Assay

    Quantification of SIV DNA and RNA in adipose tissue cells. (A, B) Quantification of SIV DNA (A) and RNA (B) was performed on SVF (n = 8), sorted adipose CD4 + T cells (CD4) or CD14-expressing cells (CD14) (n = 5) and PBMCs (n = 8) from SIV-infected animals. SIV DNA and RNA assays were performed in duplicate and the results are expressed in log SIV DNA copies per million cells. Data are quoted as the median [interquartile range]. Significant differences in a Mann-Whitney non-parametric test are shown as * p
    Figure Legend Snippet: Quantification of SIV DNA and RNA in adipose tissue cells. (A, B) Quantification of SIV DNA (A) and RNA (B) was performed on SVF (n = 8), sorted adipose CD4 + T cells (CD4) or CD14-expressing cells (CD14) (n = 5) and PBMCs (n = 8) from SIV-infected animals. SIV DNA and RNA assays were performed in duplicate and the results are expressed in log SIV DNA copies per million cells. Data are quoted as the median [interquartile range]. Significant differences in a Mann-Whitney non-parametric test are shown as * p

    Techniques Used: Expressing, Infection, MANN-WHITNEY

    19) Product Images from "Isolation and initial propagation of guinea pig adenovirus (GPAdV) inCavia porcellus cell lines"

    Article Title: Isolation and initial propagation of guinea pig adenovirus (GPAdV) inCavia porcellus cell lines

    Journal: F1000Research

    doi: 10.12688/f1000research.20135.2

    Detection of GPAdV DNA in infected tissues and cell cultures. Total DNA was extracted from GP lung homogenate, nasal scrapings or cell culture aliquots with Qiagen DNA micro kit (QIAGEN, Valencia, CA) and used as a template for PCR amplification of a portion of the hexon gene using the primers and protocol developed by Pring-Akerblom et al. (1997) . Ch-Ext. LH : chloroform-extracted lung homogenate supernatant; AUS 1 and AUS 2 : designations for two different Australian lung tissue samples; CZE NS : nasal scraping suspension from GPs experimentally infected with Czech Republic strain; C : negative control; M . EZ Load™ 100 bp Molecular Ruler (Bio-Rad, Hercules, CA); p1 : first passage in culture from original sample.
    Figure Legend Snippet: Detection of GPAdV DNA in infected tissues and cell cultures. Total DNA was extracted from GP lung homogenate, nasal scrapings or cell culture aliquots with Qiagen DNA micro kit (QIAGEN, Valencia, CA) and used as a template for PCR amplification of a portion of the hexon gene using the primers and protocol developed by Pring-Akerblom et al. (1997) . Ch-Ext. LH : chloroform-extracted lung homogenate supernatant; AUS 1 and AUS 2 : designations for two different Australian lung tissue samples; CZE NS : nasal scraping suspension from GPs experimentally infected with Czech Republic strain; C : negative control; M . EZ Load™ 100 bp Molecular Ruler (Bio-Rad, Hercules, CA); p1 : first passage in culture from original sample.

    Techniques Used: Infection, Cell Culture, Polymerase Chain Reaction, Amplification, Negative Control

    20) Product Images from "Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells"

    Article Title: Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.3003761

    Transcriptional activity and CAR function in persisting cells (A) The CD4ζ RNA level (y axis) is plotted versus the number of DNA CD4ζ DNA copies per million PBMC of each tested sample. Samples from the Deeks, Mitsuyasu and Aronson studies are plotted as red, blue and green symbols, respectively. CD4ζ RNA expression was calculated from the ΔCt values for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is expressed at a high level, so that greater expression of CD4ζ results in a smaller expression difference and so a smaller ΔCt. The values are significantly correlated by linear regression analysis (p=0.0018) testing whether rho=0 or not. No RT-controls were run in parallel and all plotted CD4ζ samples were negative, confirming the signal observed is due to RNA template. Two subjects did not have detectable CD4ζ RNA. (B) . Functionality is measured as the relative increase in the average copy number of CD4ζ cells following anti-CD4 antibody activation over percentage of CD4ζ before stimulation. (C) Fold-increase of CD4ζ expressing cells following three 10-day rounds of anti-CD4 mAb loaded irradiated K562 artificial antigen presenting cells expressing the high affinity Fc Receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ copy numbers were evaluated from the gDNA of subject PBMCs before and after activation by qPCR analysis. The final percentage of CD4ζ in each culture is indicated by the number at the top of each bar. Each bar is designated at the bottom with the subject ID and year post-infusion of the sample.
    Figure Legend Snippet: Transcriptional activity and CAR function in persisting cells (A) The CD4ζ RNA level (y axis) is plotted versus the number of DNA CD4ζ DNA copies per million PBMC of each tested sample. Samples from the Deeks, Mitsuyasu and Aronson studies are plotted as red, blue and green symbols, respectively. CD4ζ RNA expression was calculated from the ΔCt values for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is expressed at a high level, so that greater expression of CD4ζ results in a smaller expression difference and so a smaller ΔCt. The values are significantly correlated by linear regression analysis (p=0.0018) testing whether rho=0 or not. No RT-controls were run in parallel and all plotted CD4ζ samples were negative, confirming the signal observed is due to RNA template. Two subjects did not have detectable CD4ζ RNA. (B) . Functionality is measured as the relative increase in the average copy number of CD4ζ cells following anti-CD4 antibody activation over percentage of CD4ζ before stimulation. (C) Fold-increase of CD4ζ expressing cells following three 10-day rounds of anti-CD4 mAb loaded irradiated K562 artificial antigen presenting cells expressing the high affinity Fc Receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ copy numbers were evaluated from the gDNA of subject PBMCs before and after activation by qPCR analysis. The final percentage of CD4ζ in each culture is indicated by the number at the top of each bar. Each bar is designated at the bottom with the subject ID and year post-infusion of the sample.

    Techniques Used: Activity Assay, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay, Irradiation, Real-time Polymerase Chain Reaction

    21) Product Images from "Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts"

    Article Title: Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

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

    doi: 10.1073/pnas.1806305115

    Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.
    Figure Legend Snippet: Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.

    Techniques Used: Staining, Infection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    HFFs expressing Cas9 and gRNAs targeting PDGFRα survived trimer-only HCMV infection in a genome-wide CRISPR screen. Statistical analysis identified PDGFRα as the most significant hit from the screens of AD169 ( A ) and Merlin ( B ), using the MAGeCK algorithm.
    Figure Legend Snippet: HFFs expressing Cas9 and gRNAs targeting PDGFRα survived trimer-only HCMV infection in a genome-wide CRISPR screen. Statistical analysis identified PDGFRα as the most significant hit from the screens of AD169 ( A ) and Merlin ( B ), using the MAGeCK algorithm.

    Techniques Used: Expressing, Infection, Genome Wide, CRISPR

    PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.
    Figure Legend Snippet: PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.

    Techniques Used: BAC Assay, Electroporation, Immunofluorescence, Expressing, Microscopy

    IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.
    Figure Legend Snippet: IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.

    Techniques Used: Transduction, Expressing, Infection, Flow Cytometry, Cytometry, Western Blot, Real-time Polymerase Chain Reaction

    22) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    23) Product Images from "Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation"

    Article Title: Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

    Journal: Learning & Memory

    doi: 10.1101/lm.047977.118

    Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,
    Figure Legend Snippet: Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,

    Techniques Used: Methylation

    24) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    25) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    26) Product Images from "From virus isolation to metagenome generation for investigating viral diversity in deep-sea sediments"

    Article Title: From virus isolation to metagenome generation for investigating viral diversity in deep-sea sediments

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08783-4

    Comparison of viral DNA concentrations obtained using the QIAamp DNA extraction kit and the Sambrook protocol. Mean (n = 3) and standard deviations are shown. n.a. = not available.
    Figure Legend Snippet: Comparison of viral DNA concentrations obtained using the QIAamp DNA extraction kit and the Sambrook protocol. Mean (n = 3) and standard deviations are shown. n.a. = not available.

    Techniques Used: DNA Extraction

    27) Product Images from "Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation"

    Article Title: Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

    Journal: Learning & Memory

    doi: 10.1101/lm.047977.118

    Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,
    Figure Legend Snippet: Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,

    Techniques Used: Methylation

    28) Product Images from "Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation"

    Article Title: Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

    Journal: Learning & Memory

    doi: 10.1101/lm.047977.118

    Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,
    Figure Legend Snippet: Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. ( A ) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control,

    Techniques Used: Methylation

    29) Product Images from "In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure"

    Article Title: In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20080193

    Clonality and switch to IL-10–secreting T cells after bee stings. TCRVβ gene clonality was analyzed in PLA-specific IL-4–, IL-10–, and IFN-γ–secreting T cells before and 7 d after multiple bee stings. A clonal sample (C) and a nonclonal sample (N) were included as controls. The PCR products of three different test tubes were run on three different gels, and a band that results from a clonal sample appears in a range from 240 to 285 bp (primers A, tube 1), from 240 to 285 bp (primers B, tube 2), and from 285 to 325 bp and from 170 to 210 bp (primers C and D, tube 3). The specimen control size ladder master mix generates a series of amplicons to ensure that the quality and quantity of input DNA were sufficient for the test. Heteroduplex analysis of the PCR products (except for the specimen control size ladder) on 6% TBE polyacrylamide gels stained with ethidium bromide is shown. One representative out of three experiments is shown.
    Figure Legend Snippet: Clonality and switch to IL-10–secreting T cells after bee stings. TCRVβ gene clonality was analyzed in PLA-specific IL-4–, IL-10–, and IFN-γ–secreting T cells before and 7 d after multiple bee stings. A clonal sample (C) and a nonclonal sample (N) were included as controls. The PCR products of three different test tubes were run on three different gels, and a band that results from a clonal sample appears in a range from 240 to 285 bp (primers A, tube 1), from 240 to 285 bp (primers B, tube 2), and from 285 to 325 bp and from 170 to 210 bp (primers C and D, tube 3). The specimen control size ladder master mix generates a series of amplicons to ensure that the quality and quantity of input DNA were sufficient for the test. Heteroduplex analysis of the PCR products (except for the specimen control size ladder) on 6% TBE polyacrylamide gels stained with ethidium bromide is shown. One representative out of three experiments is shown.

    Techniques Used: Proximity Ligation Assay, Polymerase Chain Reaction, Staining

    30) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    31) Product Images from "Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection"

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-017-0034-3

    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Figure Legend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Techniques Used: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR
    Figure Legend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Techniques Used: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    32) Product Images from "Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling"

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0367-z

    Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining, Isolation, Sequencing

    Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Expressing, Isolation, Transfection, Clone Assay, Marker, Confocal Microscopy, Staining

    Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Expressing, Electrophoresis, Staining, Marker, Confocal Microscopy, Mouse Assay

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

    33) Product Images from "Chromatin state changes during neural development revealed by in vivo cell-type specific profiling"

    Article Title: Chromatin state changes during neural development revealed by in vivo cell-type specific profiling

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02385-4

    Profiling of in vivo chromatin states during Drosophila neural development. a Chromatin-associated proteins used in this study. Brm, HP1a and Pc are illustrated alongside the histone H3 modifications that they, respectively, associate with, together with RNA Pol II (open, permissive chromatin) and histone H1 (repressive chromatin). b GAL4 driver lines used in this study, and the cell types that they target. c , d Targeted DamID DNA-binding tracks for the chromatin proteins examined in this study are illustrated for two sample genes. c The gene dpn (an orthologue of the mammalian HES gene family) is rapidly repressed following neuronal differentiation. d The octopamine receptor gene OctbetaR1 is activated following differentiation
    Figure Legend Snippet: Profiling of in vivo chromatin states during Drosophila neural development. a Chromatin-associated proteins used in this study. Brm, HP1a and Pc are illustrated alongside the histone H3 modifications that they, respectively, associate with, together with RNA Pol II (open, permissive chromatin) and histone H1 (repressive chromatin). b GAL4 driver lines used in this study, and the cell types that they target. c , d Targeted DamID DNA-binding tracks for the chromatin proteins examined in this study are illustrated for two sample genes. c The gene dpn (an orthologue of the mammalian HES gene family) is rapidly repressed following neuronal differentiation. d The octopamine receptor gene OctbetaR1 is activated following differentiation

    Techniques Used: In Vivo, Binding Assay

    34) Product Images from "Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling"

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0367-z

    Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining, Isolation, Sequencing

    Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Expressing, Isolation, Transfection, Clone Assay, Marker, Confocal Microscopy, Staining

    Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Expressing, Electrophoresis, Staining, Marker, Confocal Microscopy, Mouse Assay

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

    35) Product Images from "Genome-wide DNA methylation analysis in precursor B-cells"

    Article Title: Genome-wide DNA methylation analysis in precursor B-cells

    Journal: Epigenetics

    doi: 10.4161/15592294.2014.983379

    Isolation of precursor B-cell subsets from human umbilical cord blood. Mononuclear cells were isolated using density gradient centrifugation to remove all non B-cells. B-cells were labeled with cell surface antibodies and sorted into 4 separate tubes. R4: pro-B-cells; R5: pre-BI cells; R6: pre-BII cells; R7: naïve B-cells.
    Figure Legend Snippet: Isolation of precursor B-cell subsets from human umbilical cord blood. Mononuclear cells were isolated using density gradient centrifugation to remove all non B-cells. B-cells were labeled with cell surface antibodies and sorted into 4 separate tubes. R4: pro-B-cells; R5: pre-BI cells; R6: pre-BII cells; R7: naïve B-cells.

    Techniques Used: Isolation, Gradient Centrifugation, Labeling

    Average read and alignment statistics. Read and alignment statistics were averaged across all individuals for each precursor B-cell subset. The top of each bar represents the total number of sequencing reads (blue), the total number of mapped reads (dark gray), and the total number of unique reads (light gray).
    Figure Legend Snippet: Average read and alignment statistics. Read and alignment statistics were averaged across all individuals for each precursor B-cell subset. The top of each bar represents the total number of sequencing reads (blue), the total number of mapped reads (dark gray), and the total number of unique reads (light gray).

    Techniques Used: Sequencing

    36) Product Images from "The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae"

    Article Title: The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01608-4

    Bayesian (using PhyloBayes 53 ) and Maximum Likelihood (using RAxML 51 ) phylogenetic analysis of mitochondrial protein-coding genes from the Metazoa, including P. rubra , I. pulchra and A. ylvae with posterior probability and bootstrap support values, respectively, at relevant nodes. Analysis carried out on trimmed alignment. Topology of both trees is identical.
    Figure Legend Snippet: Bayesian (using PhyloBayes 53 ) and Maximum Likelihood (using RAxML 51 ) phylogenetic analysis of mitochondrial protein-coding genes from the Metazoa, including P. rubra , I. pulchra and A. ylvae with posterior probability and bootstrap support values, respectively, at relevant nodes. Analysis carried out on trimmed alignment. Topology of both trees is identical.

    Techniques Used:

    Overview of the mitochondrial genome sequences we resolve for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae (Xenacoelomorpha: Acoela). Genes not drawn to scale. Numbers beneath the sequences show intergenic spaces (positive values) or intergenic overlap (negative values). Protein-coding genes are denoted by three letter abbreviations; ribosomal genes by four letter abbreviations. tRNAs are shown by single uppercase letters. ( A ) P. rubra 14,957 base-pair long sequence. All genes found on the positive (forward) strand. Where genes, rRNAs or tRNAs are coloured orange, this is solely to demonstrate overlap with adjacent genes, rRNAs or tRNAs. ( B ) I. pulchra 18,725 base-pair long sequence. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding sequence shown in grey. ( C ) A. ylvae 16,619 nucleotide-long mitochondrial genome. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding regions greater than 100 nucleotides in length are shown in grey.
    Figure Legend Snippet: Overview of the mitochondrial genome sequences we resolve for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae (Xenacoelomorpha: Acoela). Genes not drawn to scale. Numbers beneath the sequences show intergenic spaces (positive values) or intergenic overlap (negative values). Protein-coding genes are denoted by three letter abbreviations; ribosomal genes by four letter abbreviations. tRNAs are shown by single uppercase letters. ( A ) P. rubra 14,957 base-pair long sequence. All genes found on the positive (forward) strand. Where genes, rRNAs or tRNAs are coloured orange, this is solely to demonstrate overlap with adjacent genes, rRNAs or tRNAs. ( B ) I. pulchra 18,725 base-pair long sequence. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding sequence shown in grey. ( C ) A. ylvae 16,619 nucleotide-long mitochondrial genome. Genes found on the positive (forward) strand are coloured blue; genes on the negative (reverse) strand are coloured purple. Non-coding regions greater than 100 nucleotides in length are shown in grey.

    Techniques Used: Sequencing

    Comparisons of gene orders in the mitochondrial genome sequences resolved for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae compared to a published P. rubra fragment; the acoel Symsagittifera roscoffensis ; the xenoturbellid Xenoturbella bocki ; the nemertodermatid Nemertoderma westbladi and the metazoan mitochondrial ‘ground plan’ gene order, represented by Limulus polyphemus . Genes are not drawn to scale. Coloured genes chosen to show ‘anchors’ and divergence from the ground plan gene order in other species.
    Figure Legend Snippet: Comparisons of gene orders in the mitochondrial genome sequences resolved for Paratomella rubra , Isodiametra pulchra and Archaphanostoma ylvae compared to a published P. rubra fragment; the acoel Symsagittifera roscoffensis ; the xenoturbellid Xenoturbella bocki ; the nemertodermatid Nemertoderma westbladi and the metazoan mitochondrial ‘ground plan’ gene order, represented by Limulus polyphemus . Genes are not drawn to scale. Coloured genes chosen to show ‘anchors’ and divergence from the ground plan gene order in other species.

    Techniques Used:

    37) Product Images from "Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis"

    Article Title: Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddx140

    All HLHS patients harbour deleterious variants in genes involved in the NOTCH signalling pathway. ( A ) Summary of NOTCH receptor variants identified in HLHS patients. Published NOTCH variants associated with HLHS are shown in cyan, published variants associated with congenital heart disease are shown in green and published variants not associated with heart disease are shown in red. Novel variants uncovered by our exome analysis and not reported to date are shown in black font. ( B ) Confirmation of variants by direct sequencing in HLHS patient and unaffected controls (representative examples). These mutations were not present in the controls or other HLHS samples.
    Figure Legend Snippet: All HLHS patients harbour deleterious variants in genes involved in the NOTCH signalling pathway. ( A ) Summary of NOTCH receptor variants identified in HLHS patients. Published NOTCH variants associated with HLHS are shown in cyan, published variants associated with congenital heart disease are shown in green and published variants not associated with heart disease are shown in red. Novel variants uncovered by our exome analysis and not reported to date are shown in black font. ( B ) Confirmation of variants by direct sequencing in HLHS patient and unaffected controls (representative examples). These mutations were not present in the controls or other HLHS samples.

    Techniques Used: Sequencing

    38) Product Images from "Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts"

    Article Title: Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

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

    doi: 10.1073/pnas.1806305115

    Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.
    Figure Legend Snippet: Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.

    Techniques Used: Staining, Infection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.
    Figure Legend Snippet: IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.

    Techniques Used: Transduction, Expressing, Infection, Flow Cytometry, Cytometry, Western Blot, Real-time Polymerase Chain Reaction

    39) Product Images from "Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts"

    Article Title: Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

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

    doi: 10.1073/pnas.1806305115

    Pentamer-containing TB40/E raised from ARPE-19 epithelial cells enters PDGFRα-KO HFFs more efficiently than TB40/E grown from fibroblasts. ( A ) HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with TB40/E-GFP produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively. ( B ) Western blot analysis of virions produced from MRC-5 cells infected by TB40/E-fibro or TB40/E-epi for gH/gL complexes (nonreducing conditions) using anti-gH antibody and for viral major capsid protein (MCP; reducing conditions) serving as loading control for virion proteins.
    Figure Legend Snippet: Pentamer-containing TB40/E raised from ARPE-19 epithelial cells enters PDGFRα-KO HFFs more efficiently than TB40/E grown from fibroblasts. ( A ) HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with TB40/E-GFP produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively. ( B ) Western blot analysis of virions produced from MRC-5 cells infected by TB40/E-fibro or TB40/E-epi for gH/gL complexes (nonreducing conditions) using anti-gH antibody and for viral major capsid protein (MCP; reducing conditions) serving as loading control for virion proteins.

    Techniques Used: Clone Assay, Infection, Produced, Expressing, Flow Cytometry, Cytometry, Western Blot

    Virus containing pentamer enters PDGFRα-KO HFF cells using an alternative route(s). HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with viruses produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells, except for Merlin-GFP-epi and Merlin-GFP-fibro, where IE1 expression was measured to assay percentage of infected cells due to the dim GFP signal. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively.
    Figure Legend Snippet: Virus containing pentamer enters PDGFRα-KO HFF cells using an alternative route(s). HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with viruses produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells, except for Merlin-GFP-epi and Merlin-GFP-fibro, where IE1 expression was measured to assay percentage of infected cells due to the dim GFP signal. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively.

    Techniques Used: Clone Assay, Infection, Produced, Expressing, Flow Cytometry, Cytometry

    Trimer-only HCMV cannot initiate infection in PDGFRα-KO HFF cells. ( A ) Single-cell cloning of HFFs transduced with lentivirus expressing Cas9 and gRNAs against PDGFRα generated three PDGFRα-KO clones from three independent gRNAs. HFFs (CN-9) that underwent the same single-cell cloning process served as an aged control. Surface staining of PDGFRα and PDGFRβ was performed to confirm the PDGFRα-KO phenotype. ( B ) Cells in A were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and cell morphology was monitored at 5 dpi. ( C ) Cell-free virus production at 5 dpi was measured by plaque assay. Mean ± SD is presented from three biological replicates, and the red line indicates the limit of detection.
    Figure Legend Snippet: Trimer-only HCMV cannot initiate infection in PDGFRα-KO HFF cells. ( A ) Single-cell cloning of HFFs transduced with lentivirus expressing Cas9 and gRNAs against PDGFRα generated three PDGFRα-KO clones from three independent gRNAs. HFFs (CN-9) that underwent the same single-cell cloning process served as an aged control. Surface staining of PDGFRα and PDGFRβ was performed to confirm the PDGFRα-KO phenotype. ( B ) Cells in A were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and cell morphology was monitored at 5 dpi. ( C ) Cell-free virus production at 5 dpi was measured by plaque assay. Mean ± SD is presented from three biological replicates, and the red line indicates the limit of detection.

    Techniques Used: Infection, Clone Assay, Transduction, Expressing, Generated, Staining, Plaque Assay

    PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.
    Figure Legend Snippet: PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.

    Techniques Used: BAC Assay, Electroporation, Immunofluorescence, Expressing, Microscopy

    IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.
    Figure Legend Snippet: IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.

    Techniques Used: Transduction, Expressing, Infection, Flow Cytometry, Cytometry, Western Blot, Real-time Polymerase Chain Reaction

    40) Product Images from "Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling"

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0367-z

    Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of Foreign DNA sorting into EVs requires STING and TBK1. ( a, b ) Ifnb mRNA levels in Wt MEFs treated with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs (a) infected with L.monocytogenes (n=4), or (b) transfected with DNA (2μg/ml). ( c ) Wt MEFs treated for 2h with supernatants from Wt, Sting gt/gt or Tbk1 -/- MEFs stimulated with FITC-labelled DNA (1μg/ml, 18h) were subjected to confocal microscopy for visualization of FITC. Nuclei were stained with DAPI (n=6). ( d ) Quantification of the data shown in panel c. 100 cells were evaluated per group. ( e ) Immunoblot analysis of cell lysates and EVs isolated from Wt and Sting gt /gt cells. ( f ) Representatives images of AFM of DNA extracted from EVs from Wt and STING-deficient cells left untreated or infected with L. monocytogenes . Exosomal DNA in black box. ( g ) Quantification of the data shown in panel f (n=3). ( h ) Bacterial DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h. The data are represented as mapped number of bacterial genome reads from deep sequencing analysis (n=3). ( i ) Quantification of DNA in EVs isolated from Wt and Sting gt/gt MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( j ) Merged tracks of mapped bacterial DNA reads in EV DNA from L.monocytogenes- infected MEFs. The results are shown as tracked reads versus position in the L.monocytogenes genome. The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining, Isolation, Sequencing

    Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Foreign intracellular DNA stimulates IFNβ expression in bystander cells through EVs. ( a ) Ifnb induction in BMMs stimulated for 6 h with supernatants isolated from BMMs 18 h after Lipofectamine transfection with dsDNA (1μg/ml) in the presence or absence of GW4869 (10μM) (n=3). ( b ) Immunoblot analysis of two nSmase2 -/- MEF clones targeted with two different gRNAs (1a6, 3b10). ( c ) Ifnb mRNA levels in recipient Wt MEFs treated with supernatants from Wt or nSmase2 -/- MEFs transfected with DNA (n=6). ( d ) Induction of Ifnb mRNA in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells upon stimulation with supernatants from Wt MEFs transfected with DNA (n=6). ( e ) Induction of Ifnb in Wt, Mavs -/- , Sting gt/gt and Tbk1 -/- MEFs upon stimulation with supernatant from cells transfected with poly(I:C) (1 μg/ml) (n=4). ( f ) Ifnb mRNA induction in MEFs stimulated with supernatants from donor cells transfected with dsDNA of the shown sizes (n=4). ( g ) Cellular lysates and isolated EVs were analyzed by Immunoblotting for exosomal markers CD81 and CD63, and the ER marker calnexin. The EVs were also subjected to Nanoparticle Tracking Analysis for evaluation of size distribution. The Red error bars indicate one standard error of the mean (+/-), while the black curve represents the mean of three independent measurements. ( h ) Induction of Ifnb mRNA in Wt MEFs stimulated with EVs from dsDNA- or mock transfected MEFs (+/- GW4869), and with the remaining supernatant from the EV isolation procedure (n=4). ( i ) PMA-differentiated THP1 cells treated for 4 hours with supernatants from Wt MEFs stimulated with FITC-labelled DNA were subjected to confocal microscopy for visualization of FITC and IRF3. Nuclei were stained with DAPI. Merge includes the bright field image. The presented data are representative of at least 3 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Expressing, Isolation, Transfection, Clone Assay, Marker, Confocal Microscopy, Staining

    Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Listeria infection activates EV dependent stimulation of type I IFN expression in bystander cells. ( a ) Ifnb mRNA levels in Wt, Mavs -/- , cGas -/- , Sting gt/gt and Tbk1 -/- cells stimulated for 6h with supernatants from MEFs infected with L. monocytogenes (MOI 200) for 18 h (n=5). ( b ) Induction of Ifnb in MEFs (n=4) and (c) IL1β in the supernatants of BMMs (n=3) stimulated with supernatants from L. monocytogenes -infected MEFs in the presence or absence of GW4869 (10μM). ( d ) DNA extracted from EVs from supernatants of MEFs infected with L. monocytogenes (MOI 200) were analyzed by AFM. Circles are shown around extended structures with a width and height similar to DNA. Scale bar 500nm. The boxed part of the image is magnified in the image to the right for measurement of the height of exosomal DNA (~1.5 nm). ( e ) Fragment analyzer electrophoresis of DNA extracted from EVs from supernatants of MEFs infected with L.monocytogenes . Markers, 1 bp (left) and 100,000 bp (right). ( f ) Supernatants from Wt MEFs infected with EdC-labelled L. monocytogenes for 18 h, were transferred to recipient cells for 2h. The cells were stained for the early endosome marker Rab7 and EdC-labelled bacterial DNA was visualized using Click-it chemistry. The cells were analyzed by confocal microscopy. Nuclei were stained with DAPI. The graph to the right represents quantification of cells with positive fluorescent signal. For each treatment, more than 200 cells were examined (blinded). ( g ) Induction of Ifnb mRNA in MEFs infected with L. monocytogenes for 6h or 18h or treated for 6h with Gentamicin-treated supernatant from L. monocytogenes -infected MEFs (n=4). ( h ) Induction of Ifnb mRNA in MEFs infected with F.tularensis for 24h or treated for 6h with Gentamicin-treated supernatant from F.tularensis -infected MEFs (n=3). ( i, j ) Ifnb and Tnfa mRNA levels in spleens of mice left untreated or infected with L. monocytogenes (1x10 6 cfu) for 24 h in the presence of GW4869 (GW, 0.125 μg per gram bodyweight) (n=5 mice). The presented data are representative of at least 3 independent experiments. The Ifnb and Tnfa mRNA levels were normalized to bactin mRNA levels and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Infection, Expressing, Electrophoresis, Staining, Marker, Confocal Microscopy, Mouse Assay

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.
    Figure Legend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Techniques Used: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

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    Qiagen qiaamp micro kit
    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen <t>QIAamp</t> Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when <t>DNA</t> amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs
    Qiaamp Micro Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Journal: NPJ Genomic Medicine

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    doi: 10.1038/s41525-017-0034-3

    Figure Lengend Snippet: Workflow optimization for biopsy genomic profiling, tissue and CTCs. Qiagen QIAamp Micro Kit and GeneRead DNAseq targeted panel sequencing were applied on all types of cell samples. This overall workflow performs well when DNA amount is sufficient, i.e., from whole blood and tissue biopsy. For rare cells, DNA input is insufficient for targeted NGS, leading to a bias and the need for an extra step of Whole Genome Amplification (WGA). Two WGA kits were evaluated (WGA4 from Sigma and REPLI-g from Qiagen) and obtained low coverage for fixed cells, while REPLI-g was identified as optimal for fresh rare cells and used for a final validation on patient CTCs

    Article Snippet: To extract DNA from fresh cells , the QIAamp Micro Kit (Qiagen) was used following the standard cell protocol from Qiagen.

    Techniques: Sequencing, Next-Generation Sequencing, Whole Genome Amplification

    a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Journal: NPJ Genomic Medicine

    Article Title: Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

    doi: 10.1038/s41525-017-0034-3

    Figure Lengend Snippet: a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR

    Article Snippet: To extract DNA from fresh cells , the QIAamp Micro Kit (Qiagen) was used following the standard cell protocol from Qiagen.

    Techniques: DNA Extraction, Incubation, Staining, Real-time Polymerase Chain Reaction

    Using the DNA purified by the adapted AllPrep DNA/RNA method for genome profiling. A: Amplified tumor DNA fragments from DNA purified by the adapted AllPrep DNA/RNA method and by the QIAamp DNA purification method as revealed on a 2% agarose gel.

    Journal: The Journal of Molecular Diagnostics : JMD

    Article Title: Simultaneous Isolation of DNA and RNA from the Same Cell Population Obtained by Laser Capture Microdissection for Genome and Transcriptome Profiling

    doi: 10.2353/jmoldx.2008.070131

    Figure Lengend Snippet: Using the DNA purified by the adapted AllPrep DNA/RNA method for genome profiling. A: Amplified tumor DNA fragments from DNA purified by the adapted AllPrep DNA/RNA method and by the QIAamp DNA purification method as revealed on a 2% agarose gel.

    Article Snippet: For comparison, DNA and RNA were purified separately from LCM-harvested cancer cells from adjacent sections of the same tissue specimen using QIAamp DNA Micro kit (Qiagen) and PicoPure RNA Isolation kit (Molecular Devices), respectively, following the manufacturer's protocols.

    Techniques: Purification, Amplification, DNA Purification, Agarose Gel Electrophoresis

    Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER (□) and QIAamp tissue kit (▿) DNA extraction methods, and 5 μl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average ΔRQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The ΔRQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean ( n = 3).

    Journal: Applied and Environmental Microbiology

    Article Title: PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5? Nuclease (TaqMan) Assay †

    doi:

    Figure Lengend Snippet: Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER (□) and QIAamp tissue kit (▿) DNA extraction methods, and 5 μl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average ΔRQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The ΔRQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean ( n = 3).

    Article Snippet: The efficiency of individual probes was determined by PCR using purified DNA templates (QIAamp tissue kit; Qiagen, Inc., Chatsworth, Calif.) from reference strains of E. coli and other bacteria.

    Techniques: Nuclease Assay, Incubation, DNA Extraction, Amplification, Sequencing, Standard Deviation