gfp negative facs  (Qiagen)


Bioz Verified Symbol Qiagen is a verified supplier
Bioz Manufacturer Symbol Qiagen manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Name:
    RNeasy Plus Micro Kit
    Description:
    For purification of up to 45 µg total RNA from cells tissues using gDNA Eliminator columns Kit contents Qiagen RNeasy Plus Micro Kit 50 preps 14L Elution Volume 5mg Tissue Amount Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes RNeasy MinElute Spin Columns gDNA Eliminator Spin Columns Collection Tubes Carrier RNA RNase free Water and Buffers Benefits Unique gDNA Eliminator columns avoid the need for DNase Efficient removal of genomic DNA Highly reproducible yields of RNA in minutes High performance RNA for sensitive application
    Catalog Number:
    74034
    Price:
    436
    Category:
    RNeasy Plus Micro Kit
    Buy from Supplier


    Structured Review

    Qiagen gfp negative facs
    RNeasy Plus Micro Kit
    For purification of up to 45 µg total RNA from cells tissues using gDNA Eliminator columns Kit contents Qiagen RNeasy Plus Micro Kit 50 preps 14L Elution Volume 5mg Tissue Amount Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes RNeasy MinElute Spin Columns gDNA Eliminator Spin Columns Collection Tubes Carrier RNA RNase free Water and Buffers Benefits Unique gDNA Eliminator columns avoid the need for DNase Efficient removal of genomic DNA Highly reproducible yields of RNA in minutes High performance RNA for sensitive application
    https://www.bioz.com/result/gfp negative facs/product/Qiagen
    Average 93 stars, based on 35818 article reviews
    Price from $9.99 to $1999.99
    gfp negative facs - by Bioz Stars, 2020-04
    93/100 stars

    Images

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

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

    Journal: eLife

    doi: 10.7554/eLife.36187

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

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

    2) Product Images from "Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human"

    Article Title: Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002071

    ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.
    Figure Legend Snippet: ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Methylation, Mouse Assay, Modification, Real-time Polymerase Chain Reaction, Mutagenesis, Derivative Assay

    Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.
    Figure Legend Snippet: Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, In Situ Hybridization

    RNA–seq profiles of control and HoxC mutant mice. RNA was extracted from the region enriched in mHotair transcripts at day 13.5, i.e. the posterior part of the fetus, including the tail, hindlimbs and the outgrowing genitalia. Plotted are mean values of 25 bp windows. (A) Transcription profiles of the four different Hox gene clusters. The positions of the genes are indicated below. (A) Expression profiles of all four Hox loci, shown with the orientation with respect to the centromers. The strong peak in the deleted HoxC cluster is a transcript induced over the second exon of Hoxc4 (non-deleted) after deletion of the cluster (see the text). (B) Examples of transcriptional variations induced by the deletion of the HoxC cluster, with some genes being slightly up-regulated ( Hoxa7 , Hoxb9 , Hoxd10 and Wsb1 ), some being down-regulated ( Igf2r , Slc15a2 , Asb4 ). Hoxd13 is shown as an unaffected control gene ( Hoxd13 ). (C) Percentage of genes either up-regulated or down-regulated in HoxC mutant animals, which were also reported to be the targets of SUZ12 in ES cells. The percentages are comparable, suggesting that capacity to recruit PRC2 may not be the main cause of the transcriptional variations observed in the HoxC mutant animals, in these tissues at this developmental time. (D) Absolute quantifications of posterior Hoxd gene transcripts and of mHotair in posterior parts of fetuses including the hindlimbs, the genital bud and the developing tail of E11.5 embryos. All values are normalized to a housekeeping gene.
    Figure Legend Snippet: RNA–seq profiles of control and HoxC mutant mice. RNA was extracted from the region enriched in mHotair transcripts at day 13.5, i.e. the posterior part of the fetus, including the tail, hindlimbs and the outgrowing genitalia. Plotted are mean values of 25 bp windows. (A) Transcription profiles of the four different Hox gene clusters. The positions of the genes are indicated below. (A) Expression profiles of all four Hox loci, shown with the orientation with respect to the centromers. The strong peak in the deleted HoxC cluster is a transcript induced over the second exon of Hoxc4 (non-deleted) after deletion of the cluster (see the text). (B) Examples of transcriptional variations induced by the deletion of the HoxC cluster, with some genes being slightly up-regulated ( Hoxa7 , Hoxb9 , Hoxd10 and Wsb1 ), some being down-regulated ( Igf2r , Slc15a2 , Asb4 ). Hoxd13 is shown as an unaffected control gene ( Hoxd13 ). (C) Percentage of genes either up-regulated or down-regulated in HoxC mutant animals, which were also reported to be the targets of SUZ12 in ES cells. The percentages are comparable, suggesting that capacity to recruit PRC2 may not be the main cause of the transcriptional variations observed in the HoxC mutant animals, in these tissues at this developmental time. (D) Absolute quantifications of posterior Hoxd gene transcripts and of mHotair in posterior parts of fetuses including the hindlimbs, the genital bud and the developing tail of E11.5 embryos. All values are normalized to a housekeeping gene.

    Techniques Used: RNA Sequencing Assay, Mutagenesis, Mouse Assay, Expressing

    3) Product Images from "Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human"

    Article Title: Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002071

    ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.
    Figure Legend Snippet: ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Methylation, Mouse Assay, Modification, Real-time Polymerase Chain Reaction, Mutagenesis, Derivative Assay

    Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.
    Figure Legend Snippet: Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, In Situ Hybridization

    4) Product Images from "Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip"

    Article Title: Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.6b12236

    Characterization of the patient-derived breast (Brx) CTC line using imaging flow cytometry. Data compare viability, EpCAM expression, and area of control versus captured/released cells from our NP- HB CTC-Chip. Representative images of one viable, cluster, and dead Brx cell (A) obtained from culture (control) and (B) captured/released from our microfluidic device. Gate settings of (C) control and (D) captured/released Brx cells. Viable cells are defined as calcein positive and caspase 3/7 negative, whereas dead cells are caspase 3/7 positive. The intensity of EpCAM obtained from (E) control and (F) captured/released Brx cells. The area of (G) control and (H) captured/released Brx cells. (I) Heat map of the C t values of seven genes obtained by RT-qPCR. Comparisons were across control, released Brx cells, and white blood cells (WBC).
    Figure Legend Snippet: Characterization of the patient-derived breast (Brx) CTC line using imaging flow cytometry. Data compare viability, EpCAM expression, and area of control versus captured/released cells from our NP- HB CTC-Chip. Representative images of one viable, cluster, and dead Brx cell (A) obtained from culture (control) and (B) captured/released from our microfluidic device. Gate settings of (C) control and (D) captured/released Brx cells. Viable cells are defined as calcein positive and caspase 3/7 negative, whereas dead cells are caspase 3/7 positive. The intensity of EpCAM obtained from (E) control and (F) captured/released Brx cells. The area of (G) control and (H) captured/released Brx cells. (I) Heat map of the C t values of seven genes obtained by RT-qPCR. Comparisons were across control, released Brx cells, and white blood cells (WBC).

    Techniques Used: Derivative Assay, Imaging, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

    5) Product Images from "Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability"

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability

    Journal: Neural Development

    doi: 10.1186/1749-8104-9-19

    Islet1 knock-down affects sensory neuron differentiation. (A-C) In control embryos (A, B) , Rohon-Beard cells (RBs) express runx3 . Islet1 knock-down leads to fewer cells with robust expression of runx3 (C). The asterisk denotes a cell expressing the gene. (D-F’) Tg(-3.4neurog1:gfp)sb4 control (D-E’) and E3 morphant (F, F’) 24 hours post-fertilization (hpf) embryos were examined for expression of olig4 (red). (D-E’) In lateral views of the dorsal spinal cord, RNA in situ hybridization reveals expression of the interneuron marker olig4 (red). Green fluorescent protein (GFP) + neurons do not express olig4 and comprise RBs and dorsal lateral ascending interneurons (see Figure 2 ). (F-F’) Islet1 knock-down leads to an increase in the number of olig4 expressing cells within the dorsal spinal cord. However, similar to RBs of control embryos (D-E’) , RB-like neurons do not express detectable levels of olig4 (F) . (G-I) At 72 hpf, dorsal root ganglia (DRGs) are easily identified as GFP + neurons with large somata located near the spinal cord/notochord border. (G, H) In control embryos, DRG neurons project from their soma bipolar axons that extend dorsally and ventrally (asterisks). (I) Islet1 knock-down reduces the number of GFP + DRGs. Furthermore, for the few GFP + DRGs remaining, their axons show abnormal morphologies. Scale bars = 50 μm in A (for A-C ), D (for D-F ’) and G (for G-I ). CtlMO, 5-base mismatched; islet1 (Sp)E3MO; E3MO, E3 morpholino; Uninj, uninjected.
    Figure Legend Snippet: Islet1 knock-down affects sensory neuron differentiation. (A-C) In control embryos (A, B) , Rohon-Beard cells (RBs) express runx3 . Islet1 knock-down leads to fewer cells with robust expression of runx3 (C). The asterisk denotes a cell expressing the gene. (D-F’) Tg(-3.4neurog1:gfp)sb4 control (D-E’) and E3 morphant (F, F’) 24 hours post-fertilization (hpf) embryos were examined for expression of olig4 (red). (D-E’) In lateral views of the dorsal spinal cord, RNA in situ hybridization reveals expression of the interneuron marker olig4 (red). Green fluorescent protein (GFP) + neurons do not express olig4 and comprise RBs and dorsal lateral ascending interneurons (see Figure 2 ). (F-F’) Islet1 knock-down leads to an increase in the number of olig4 expressing cells within the dorsal spinal cord. However, similar to RBs of control embryos (D-E’) , RB-like neurons do not express detectable levels of olig4 (F) . (G-I) At 72 hpf, dorsal root ganglia (DRGs) are easily identified as GFP + neurons with large somata located near the spinal cord/notochord border. (G, H) In control embryos, DRG neurons project from their soma bipolar axons that extend dorsally and ventrally (asterisks). (I) Islet1 knock-down reduces the number of GFP + DRGs. Furthermore, for the few GFP + DRGs remaining, their axons show abnormal morphologies. Scale bars = 50 μm in A (for A-C ), D (for D-F ’) and G (for G-I ). CtlMO, 5-base mismatched; islet1 (Sp)E3MO; E3MO, E3 morpholino; Uninj, uninjected.

    Techniques Used: Expressing, RNA In Situ Hybridization, Marker

    6) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    7) Product Images from "Inhibition of the apelin/apelin receptor axis decreases cholangiocarcinoma growth"

    Article Title: Inhibition of the apelin/apelin receptor axis decreases cholangiocarcinoma growth

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2016.11.025

    A: Positive APLNR staining by IHC in four human CCA tissues (bottom row) compared to adjacent non-malignant liver tissues (top row). Semiquantitative analysis of IHC images shows significantly increased expression of APLNR in CCA tissues compare to non-malignant liver tissues (n = 18). Table 1: Corresponding patient demographics and tumor grade from patients included in IHC images. B: APLNR gene expression is increased by rtPCR in human CCA tissues (n = 3). Patient samples are labeled as G = tumor grade, followed by the patient age. C: Apelin gene expression is increased by rtPCR in human CCA tissues (n = 3). Patient samples are labeled as G = tumor grade, followed by the patient age (* = P
    Figure Legend Snippet: A: Positive APLNR staining by IHC in four human CCA tissues (bottom row) compared to adjacent non-malignant liver tissues (top row). Semiquantitative analysis of IHC images shows significantly increased expression of APLNR in CCA tissues compare to non-malignant liver tissues (n = 18). Table 1: Corresponding patient demographics and tumor grade from patients included in IHC images. B: APLNR gene expression is increased by rtPCR in human CCA tissues (n = 3). Patient samples are labeled as G = tumor grade, followed by the patient age. C: Apelin gene expression is increased by rtPCR in human CCA tissues (n = 3). Patient samples are labeled as G = tumor grade, followed by the patient age (* = P

    Techniques Used: Staining, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling

    A: Apelin treatment promotes Mz-ChA-1 gene expression of Ki-67 and PCNA (left), whereas, ML221 treatment decreases Mz-ChA-1 gene expression of Ki-67 and PCNA in a dose dependent manner (n = 5) via rtPCR. B: Gene expression of angiogenic factors (VEGF-A, Ang-1, and Ang-2) is increased when Mz-ChA-1 cells are treated with increasing concentrations of apelin (left), whereas, gene expression is decreased with increasing concentrations of ML221, an APLNR antagonist (right) (n = 5) via rtPCR. C: 10 μM of ML221 treatment significantly decreases cell proliferation and migration at 6, 12, 24, and 48 h during wound-healing assay (* = P
    Figure Legend Snippet: A: Apelin treatment promotes Mz-ChA-1 gene expression of Ki-67 and PCNA (left), whereas, ML221 treatment decreases Mz-ChA-1 gene expression of Ki-67 and PCNA in a dose dependent manner (n = 5) via rtPCR. B: Gene expression of angiogenic factors (VEGF-A, Ang-1, and Ang-2) is increased when Mz-ChA-1 cells are treated with increasing concentrations of apelin (left), whereas, gene expression is decreased with increasing concentrations of ML221, an APLNR antagonist (right) (n = 5) via rtPCR. C: 10 μM of ML221 treatment significantly decreases cell proliferation and migration at 6, 12, 24, and 48 h during wound-healing assay (* = P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Migration, Wound Healing Assay

    8) Product Images from "A Genetic Approach to the Recruitment of PRC2 at the HoxD Locus"

    Article Title: A Genetic Approach to the Recruitment of PRC2 at the HoxD Locus

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003951

    Effect of large deletions upon the H3K27me3 profiles. (A) Wild type genomic landscape of the murine HoxD cluster. The log2 profiles of H3K27me3 enrichment are depicted in red. (B–C) H3K27me3 profiles of either wild type, or animals harboring a deletion of the 5′ border of the H3K27me3 domain. Genotypes are specified on the left. (D) H3K27me3 profiles over the endogenous (left) and transgenic (right) Hoxd10 sequence and over the integration site, in the presence or absence of the transgene. Wild type GC density using a 500 bp sliding window is depicted by the black line (right panel).
    Figure Legend Snippet: Effect of large deletions upon the H3K27me3 profiles. (A) Wild type genomic landscape of the murine HoxD cluster. The log2 profiles of H3K27me3 enrichment are depicted in red. (B–C) H3K27me3 profiles of either wild type, or animals harboring a deletion of the 5′ border of the H3K27me3 domain. Genotypes are specified on the left. (D) H3K27me3 profiles over the endogenous (left) and transgenic (right) Hoxd10 sequence and over the integration site, in the presence or absence of the transgene. Wild type GC density using a 500 bp sliding window is depicted by the black line (right panel).

    Techniques Used: Transgenic Assay, Sequencing

    H3K27me3 profiles of transgenic constructs in iPS cells. (A) Western blot for pluripotency markers and morphology of the iPS del(Hoxd10) clone. (B) Chromatin signature of iPS cells with the observed reactivation of bivalent domains over most Hoxd genes. (C) H3K27me3 (red) profiles of various constructs electroporated into iPS cells carrying a deletion of Hoxd10 . The electroporated construct is depicted by the white box above each profile. Env- corresponds to transgenes carrying arms for recombination, homologous to Hoxd11 and the 3′ portion of Hoxd10 , respectively. CRE indicates single copy integrants. Black line in the GC content panel corresponds to a running window of 200 bp. PREd10 overlaps with sites of DNase hypersensitivity and experimentally validated CTCF sites. (D) ChIP-qPCR of PRC1 ( Ring1B ) and PRC2 ( Suz12 ) over various regions of the HoxD cluster. Consistent with the presence of H3K27me3, PRC1/2 binds randomly integrated PREd10 at levels comparable to that found in the endogenous Hox locus ( Hoxd13 ).
    Figure Legend Snippet: H3K27me3 profiles of transgenic constructs in iPS cells. (A) Western blot for pluripotency markers and morphology of the iPS del(Hoxd10) clone. (B) Chromatin signature of iPS cells with the observed reactivation of bivalent domains over most Hoxd genes. (C) H3K27me3 (red) profiles of various constructs electroporated into iPS cells carrying a deletion of Hoxd10 . The electroporated construct is depicted by the white box above each profile. Env- corresponds to transgenes carrying arms for recombination, homologous to Hoxd11 and the 3′ portion of Hoxd10 , respectively. CRE indicates single copy integrants. Black line in the GC content panel corresponds to a running window of 200 bp. PREd10 overlaps with sites of DNase hypersensitivity and experimentally validated CTCF sites. (D) ChIP-qPCR of PRC1 ( Ring1B ) and PRC2 ( Suz12 ) over various regions of the HoxD cluster. Consistent with the presence of H3K27me3, PRC1/2 binds randomly integrated PREd10 at levels comparable to that found in the endogenous Hox locus ( Hoxd13 ).

    Techniques Used: Transgenic Assay, Construct, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    H3K27me3 profiles on transgenes in embryo . (A) Experimental setup, with a portions of the Hoxd10 region injected into mice harboring a deletion of Hoxd10 such as to distinguish between the methylation covering the endogenous locus (light red) and the transgene (dark red). (B) H3K27me3 profiles of wild type and animals carrying a transgene corresponding to the entire part -or portions thereof- of the DNA segment deleted in the del(10) allele. Methylation over the transgenic construct(s) is depicted in dark red. CpG islands are shown as green boxes. The orange box in the WT profile indicates the position of the probe for Southern blot in which PvuII (P) and HindIII (H) were used for digestion (see figure S3 ).
    Figure Legend Snippet: H3K27me3 profiles on transgenes in embryo . (A) Experimental setup, with a portions of the Hoxd10 region injected into mice harboring a deletion of Hoxd10 such as to distinguish between the methylation covering the endogenous locus (light red) and the transgene (dark red). (B) H3K27me3 profiles of wild type and animals carrying a transgene corresponding to the entire part -or portions thereof- of the DNA segment deleted in the del(10) allele. Methylation over the transgenic construct(s) is depicted in dark red. CpG islands are shown as green boxes. The orange box in the WT profile indicates the position of the probe for Southern blot in which PvuII (P) and HindIII (H) were used for digestion (see figure S3 ).

    Techniques Used: Injection, Mouse Assay, Methylation, Transgenic Assay, Construct, Southern Blot

    9) Product Images from "Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells"

    Article Title: Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084072

    Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.
    Figure Legend Snippet: Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.

    Techniques Used: Expressing, Microarray

    10) Product Images from "Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates"

    Article Title: Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41505-6

    Differential expression of transcripts from RNA-SEQ. ( a – d ) Volcano plots comparing fold-change and p-value of each identified transcript accession for ( a , b ) U87 mg and ( c , d ) DAOY cellular aggregates for samples exposed to 250 V/m dcEFs for ( a , c ) 2 h, or ( b , d ) 8 h compared 0 h, unexposed controls. Significance threshold is set to FDR
    Figure Legend Snippet: Differential expression of transcripts from RNA-SEQ. ( a – d ) Volcano plots comparing fold-change and p-value of each identified transcript accession for ( a , b ) U87 mg and ( c , d ) DAOY cellular aggregates for samples exposed to 250 V/m dcEFs for ( a , c ) 2 h, or ( b , d ) 8 h compared 0 h, unexposed controls. Significance threshold is set to FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    11) Product Images from "Deconstructing the principles of ductal network formation in the pancreas"

    Article Title: Deconstructing the principles of ductal network formation in the pancreas

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2002842

    Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.
    Figure Legend Snippet: Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.

    Techniques Used: Cell Culture, In Vitro, Generated

    In silico network creation of ventral E12.5 networks. (A) Schematic of the in silico model. Step I: A node is created in the neighborhood of the existing network. Step II: The created node links to M nodes drawn from a pool of the nearest M + Δ nodes. Step III: The model reiterates until the desired node amount has been reached. (B) Network evolution of the in silico model from 2 nodes to 320 nodes. (C) Distribution of polygonal features for the in silico network nodes and the E12.5 pancreas network. Error bars represent SEM. Code files “NetworkCreation”, “ConvertToAdjMat”, “ConvertToAdjList”, “PlotNetwork”, “NetworkShapes”, “NetworkProp”, “FindTriangles”,”Remove_kinks” are provided in supporting information ( S1 Data ). E, embryonic day; ns, not significant; VP, ventral pancreas.
    Figure Legend Snippet: In silico network creation of ventral E12.5 networks. (A) Schematic of the in silico model. Step I: A node is created in the neighborhood of the existing network. Step II: The created node links to M nodes drawn from a pool of the nearest M + Δ nodes. Step III: The model reiterates until the desired node amount has been reached. (B) Network evolution of the in silico model from 2 nodes to 320 nodes. (C) Distribution of polygonal features for the in silico network nodes and the E12.5 pancreas network. Error bars represent SEM. Code files “NetworkCreation”, “ConvertToAdjMat”, “ConvertToAdjList”, “PlotNetwork”, “NetworkShapes”, “NetworkProp”, “FindTriangles”,”Remove_kinks” are provided in supporting information ( S1 Data ). E, embryonic day; ns, not significant; VP, ventral pancreas.

    Techniques Used: In Silico

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

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

    Journal: eLife

    doi: 10.7554/eLife.36187

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

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

    13) Product Images from "A genome‐wide si RNA screen for regulators of tumor suppressor p53 activity in human non‐small cell lung cancer cells identifies components of the RNA splicing machinery as targets for anticancer treatment"

    Article Title: A genome‐wide si RNA screen for regulators of tumor suppressor p53 activity in human non‐small cell lung cancer cells identifies components of the RNA splicing machinery as targets for anticancer treatment

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12052

    Analysis of alternative splicing of TP 53 , MDM 2 , and MDM 4 upon splice factor knockdown. A549 cells were transfected with si NT #1, si SF 3B1, si SF 3B6, si SNRPD 3, or si SF 3A3, and mRNA expression was quantified by qRT ‐ PCR . (A) Splice factor gene knockdown efficiency. For each splice factor gene, relative expression in specific knockdown cells versus si NT #1 transfected cells is shown. Panels B‐D show absolute expression normalized to β‐actin expression of TP 53 transcript variants (B), MDM 2 transcript variants (C), and MDM 4 transcript variants (D). Data are the means of three independent experiments. Error bars represent standard deviation. Significance of differential expression compared to nontargeting si RNA ‐transfected cells was tested using Student's t ‐test. * P
    Figure Legend Snippet: Analysis of alternative splicing of TP 53 , MDM 2 , and MDM 4 upon splice factor knockdown. A549 cells were transfected with si NT #1, si SF 3B1, si SF 3B6, si SNRPD 3, or si SF 3A3, and mRNA expression was quantified by qRT ‐ PCR . (A) Splice factor gene knockdown efficiency. For each splice factor gene, relative expression in specific knockdown cells versus si NT #1 transfected cells is shown. Panels B‐D show absolute expression normalized to β‐actin expression of TP 53 transcript variants (B), MDM 2 transcript variants (C), and MDM 4 transcript variants (D). Data are the means of three independent experiments. Error bars represent standard deviation. Significance of differential expression compared to nontargeting si RNA ‐transfected cells was tested using Student's t ‐test. * P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    14) Product Images from "A susceptibility locus rs7099208 is associated with non-obstructive azoospermia via reduction in the expression of FAM160B1"

    Article Title: A susceptibility locus rs7099208 is associated with non-obstructive azoospermia via reduction in the expression of FAM160B1

    Journal: Journal of Biomedical Research

    doi: 10.7555/JBR.29.20150034

    Through eQTL analysis SNP rs7099208 with gene from the Genotype-Tissue Expression project (GTEx, http://commonfund.nih.gov/GTEx/index ). SNP rs7099208 existed eQTL with gene FAM160B1 (A), P = 0.05, instead of the other two: ABLIM1 (B) and TRUB1 (C). D: This eQTL was tissue-specific, existed only in the testis, while outside of comprehensive blood.
    Figure Legend Snippet: Through eQTL analysis SNP rs7099208 with gene from the Genotype-Tissue Expression project (GTEx, http://commonfund.nih.gov/GTEx/index ). SNP rs7099208 existed eQTL with gene FAM160B1 (A), P = 0.05, instead of the other two: ABLIM1 (B) and TRUB1 (C). D: This eQTL was tissue-specific, existed only in the testis, while outside of comprehensive blood.

    Techniques Used: Expressing

    FAM160B1 gene expression in Human. A: The expression of FAM160B1 in multi-organization successively: heart, liver, spleen, lung, kidney pancreas, brain, skeletal muscle, ovarian and testes. GAPDH was used as an internal control. B-G: Immunohistochemistry of FAM160B1 in the normal Human testes. B, C, D: the negative control; E, F, G: Human testis. FAM160B1 located in post-meiotic germ cells containing spermatocytes and round spermatids.
    Figure Legend Snippet: FAM160B1 gene expression in Human. A: The expression of FAM160B1 in multi-organization successively: heart, liver, spleen, lung, kidney pancreas, brain, skeletal muscle, ovarian and testes. GAPDH was used as an internal control. B-G: Immunohistochemistry of FAM160B1 in the normal Human testes. B, C, D: the negative control; E, F, G: Human testis. FAM160B1 located in post-meiotic germ cells containing spermatocytes and round spermatids.

    Techniques Used: Expressing, Immunohistochemistry, Negative Control

    FAM160B1 gene expression in Mice. A: The expression of FAM160B1 in multi-organization successively: heart, liver, spleen, lung, kidney pancreas, brain, skeletal muscle, ovarian and testes. Actin was used as an internal control. B-D: immunohistochemistry of FAM160B1 in the mice testes. B: the negative control. C, D: FAM160B1 showed punctate signals in germ cells including spermatocytes (Spc) and round sperm (rSt), and highly expressed in elongated sperm. E-G: Expression of FAM160B1 in GC2 cells. E: the negative control. F, G: FAM160B1 located in the cytoplasm and part of the nucleus.
    Figure Legend Snippet: FAM160B1 gene expression in Mice. A: The expression of FAM160B1 in multi-organization successively: heart, liver, spleen, lung, kidney pancreas, brain, skeletal muscle, ovarian and testes. Actin was used as an internal control. B-D: immunohistochemistry of FAM160B1 in the mice testes. B: the negative control. C, D: FAM160B1 showed punctate signals in germ cells including spermatocytes (Spc) and round sperm (rSt), and highly expressed in elongated sperm. E-G: Expression of FAM160B1 in GC2 cells. E: the negative control. F, G: FAM160B1 located in the cytoplasm and part of the nucleus.

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Negative Control

    TUNEL analysis and electron microscopy after transfection with morpholinos specific to FAM160B1 . A, C: Positive signals mo-control and mo-FAM160B1 with TUNEL analysis. B, D: Larger image of A, B. E-L: Electron microscopy under morpholinos inhibiting of the two groups. E, I: the control, F, J: Larger image of E, I. G, K: in perinuclear of cells there were more lipofuscin (black arrow), lipid droplets (red arrow), vacuoles (white arrow), etc. H, L: Larger image of G, K.
    Figure Legend Snippet: TUNEL analysis and electron microscopy after transfection with morpholinos specific to FAM160B1 . A, C: Positive signals mo-control and mo-FAM160B1 with TUNEL analysis. B, D: Larger image of A, B. E-L: Electron microscopy under morpholinos inhibiting of the two groups. E, I: the control, F, J: Larger image of E, I. G, K: in perinuclear of cells there were more lipofuscin (black arrow), lipid droplets (red arrow), vacuoles (white arrow), etc. H, L: Larger image of G, K.

    Techniques Used: TUNEL Assay, Electron Microscopy, Transfection

    FAM160B1 was knocked down in murine germ cell lines GC2 with morpholinos. A, B: knockdown with splice blocking morpholinos; C: cell counting experiments between control (mo-con) and FAM160B1 (mo-fam). ** P
    Figure Legend Snippet: FAM160B1 was knocked down in murine germ cell lines GC2 with morpholinos. A, B: knockdown with splice blocking morpholinos; C: cell counting experiments between control (mo-con) and FAM160B1 (mo-fam). ** P

    Techniques Used: Blocking Assay, Cell Counting

    Immunohistochemistry of FAM160B1 in the OA testes and NOA testes. A: a-d: 4 cases of the OA. B: in spermatocyte arrest group of NOA testes (without spermatid and mature spermatozoa but contained spermatocyte). a-h: 8 cases of the spermatocyte arrest. C: in spermatid arrest group of NOA testes (without mature spermatozoa but contained round spermatid). a-d: 4 cases of the spermatid arrest. Spc: spermatocyte, rSt: round spermatid.
    Figure Legend Snippet: Immunohistochemistry of FAM160B1 in the OA testes and NOA testes. A: a-d: 4 cases of the OA. B: in spermatocyte arrest group of NOA testes (without spermatid and mature spermatozoa but contained spermatocyte). a-h: 8 cases of the spermatocyte arrest. C: in spermatid arrest group of NOA testes (without mature spermatozoa but contained round spermatid). a-d: 4 cases of the spermatid arrest. Spc: spermatocyte, rSt: round spermatid.

    Techniques Used: Immunohistochemistry

    15) Product Images from "Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes"

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5608-2

    Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM
    Figure Legend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Techniques Used: Lysis, Isolation

    Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit
    Figure Legend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Techniques Used: Lysis, Isolation, FACS

    Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P
    Figure Legend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Techniques Used: Purification, FACS, Quantitative RT-PCR, Isolation

    16) Product Images from "Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis"

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    Journal: eLife

    doi: 10.7554/eLife.40556

    let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).
    Figure Legend Snippet: let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).

    Techniques Used: Binding Assay, Reporter Assay, Sequencing, Transfection, Construct, Fluorescence, Immunostaining, Expressing, Staining

    The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.
    Figure Legend Snippet: The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.

    Techniques Used: In Vivo, Binding Assay, Activity Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Injection, Immunohistochemistry, Expressing, Plasmid Preparation, Construct, Quantitation Assay

    Effects of Lin28a loss-of-function on cell death, proliferation and the morphology of cranial ganglia. ( a–i ) Disruption of Lin28a/ let-7 axis does not cause cell death or proliferation defects in the dorsal neural tube. ( a ) Whole mount view of an HH9 embryo bilaterally transfected with control MO (blue) on the left and the Lin28a MO (green) on the right. Transverse sections showing the immunostaining for phosho-H3 (S10) ( b ) and Caspase-3 ( c ) in these embryos. ( d–e ) Transverse sections of HH9 embryos bilaterally transfected with let-7 mimic immunostained for phospho-H3 ( d ) or Caspase-3 ( e ). Quantification of average number of phosho-H3 ( f ) and Caspase-3 ( g ) positive cells on the neural tube/section in embryos transfected with Lin28a MO. Quantification of average number of phospho-H3 ( h ) and Caspase-3 positive ( i ) cells on the neural tube/section in embryos transfected with let-7 mimic. ( j–o ) Loss of Lin28a during early development disrupts the formation of neural crest derived trigeminal ganglia. Lateral view of Dapi-stained HH15 embryo showing control ( j ) and Lin28a MO ( m ) sides (control images were flipped horizontally to facilitate comparison). Immunohistochemistry for neuronal marker Tuj1 in control ( k ) and morpholino treated ( n ) side of the embryo shows the morphology of the trigeminal ganglion (white arrows point to the base of the ganglia). Higher magnification images of the trigeminal ganglia on the control ( l ) and morpholino-transfected ( o ) side of the embryo show disorganization of the trigeminal ganglia (n = 6/8). The maxillomandibular branch (red arrows) is significantly thinner in the MO transfected side. The white arrows point to the neurons of the ophthalmic branch which are disorganised on the morphants. MO: Morpholino, ns: not significant.
    Figure Legend Snippet: Effects of Lin28a loss-of-function on cell death, proliferation and the morphology of cranial ganglia. ( a–i ) Disruption of Lin28a/ let-7 axis does not cause cell death or proliferation defects in the dorsal neural tube. ( a ) Whole mount view of an HH9 embryo bilaterally transfected with control MO (blue) on the left and the Lin28a MO (green) on the right. Transverse sections showing the immunostaining for phosho-H3 (S10) ( b ) and Caspase-3 ( c ) in these embryos. ( d–e ) Transverse sections of HH9 embryos bilaterally transfected with let-7 mimic immunostained for phospho-H3 ( d ) or Caspase-3 ( e ). Quantification of average number of phosho-H3 ( f ) and Caspase-3 ( g ) positive cells on the neural tube/section in embryos transfected with Lin28a MO. Quantification of average number of phospho-H3 ( h ) and Caspase-3 positive ( i ) cells on the neural tube/section in embryos transfected with let-7 mimic. ( j–o ) Loss of Lin28a during early development disrupts the formation of neural crest derived trigeminal ganglia. Lateral view of Dapi-stained HH15 embryo showing control ( j ) and Lin28a MO ( m ) sides (control images were flipped horizontally to facilitate comparison). Immunohistochemistry for neuronal marker Tuj1 in control ( k ) and morpholino treated ( n ) side of the embryo shows the morphology of the trigeminal ganglion (white arrows point to the base of the ganglia). Higher magnification images of the trigeminal ganglia on the control ( l ) and morpholino-transfected ( o ) side of the embryo show disorganization of the trigeminal ganglia (n = 6/8). The maxillomandibular branch (red arrows) is significantly thinner in the MO transfected side. The white arrows point to the neurons of the ophthalmic branch which are disorganised on the morphants. MO: Morpholino, ns: not significant.

    Techniques Used: Transfection, Immunostaining, Derivative Assay, Staining, Immunohistochemistry, Marker

    17) Product Images from "Deconstructing the principles of ductal network formation in the pancreas"

    Article Title: Deconstructing the principles of ductal network formation in the pancreas

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2002842

    Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.
    Figure Legend Snippet: Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.

    Techniques Used: Cell Culture, In Vitro, Generated

    18) Product Images from "Deconstructing the principles of ductal network formation in the pancreas"

    Article Title: Deconstructing the principles of ductal network formation in the pancreas

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2002842

    Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.
    Figure Legend Snippet: Swelling assays reveal forskolin-induced secretion. (A) E12.5 pancreata cultured in vitro for 1 day and submitted to forskolin treatment activating the CFTR channels undergo lumen swelling. The lumen appears as a dark shadow, and some are highlighted with arrows (B). Ductal spheres generated from E12.5 pancreata swell upon treatment with forskolin, as compared to untreated controls. CFTR, cystic fibrosis transmembrane conductance regulator; E, embryonic day.

    Techniques Used: Cell Culture, In Vitro, Generated

    In silico network creation of ventral E12.5 networks. ). E, embryonic day; ns, not significant; VP, ventral pancreas.
    Figure Legend Snippet: In silico network creation of ventral E12.5 networks. ). E, embryonic day; ns, not significant; VP, ventral pancreas.

    Techniques Used: In Silico

    19) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    NAM suppresses production of AMD-related disease markers in AMD and control hiPSC-RPE (A) Percentage mRNA expression for AMD/drusen transcripts analyzed by qPCR in AMD and control hiPSC-RPE treated with 10mM NAM relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM. , lower panel). (C-F) Secretion of APOJ (C), VEGF-A (D), Aβ-40 (E) and Aβ-42 (F) measured by ELISA into the culture supernatant 60–72 hours after the last medium change in AMD and control hiPSC-RPE treated with 10mM NAM or vehicle. ; each sample is color matched across NAM and vehicle treatment. Paired Student’s t test (one-tailed) was used for statistical analysis (*= p
    Figure Legend Snippet: NAM suppresses production of AMD-related disease markers in AMD and control hiPSC-RPE (A) Percentage mRNA expression for AMD/drusen transcripts analyzed by qPCR in AMD and control hiPSC-RPE treated with 10mM NAM relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM. , lower panel). (C-F) Secretion of APOJ (C), VEGF-A (D), Aβ-40 (E) and Aβ-42 (F) measured by ELISA into the culture supernatant 60–72 hours after the last medium change in AMD and control hiPSC-RPE treated with 10mM NAM or vehicle. ; each sample is color matched across NAM and vehicle treatment. Paired Student’s t test (one-tailed) was used for statistical analysis (*= p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    hiPSC-RPE from total AMD and AMD ARMS2/HTRA1 donors express higher levels of disease-related markers compared to controls (A) Experiment schematic. (B) AMD and control hiPSC-RPE stained for Phalloidin, and the RPE markers CRALBP, OTX2 and MCT1 (Scale bar: 100µM). Insets show digitally zoomed high magnification images. (C) AMD and control hiPSC-RPE showed similar TER. (D) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for AMD/drusen transcripts. (E-F) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of Aβ peptides (E) and VEGF-A (F). (G) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for complement/inflammatory transcripts. (H) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of C3. Data are expressed as mean± SEM. Unpaired Student’s t test (one-tailed) (D–H) and two-way ANOVA (D, G) were used for statistical analysis (*= p
    Figure Legend Snippet: hiPSC-RPE from total AMD and AMD ARMS2/HTRA1 donors express higher levels of disease-related markers compared to controls (A) Experiment schematic. (B) AMD and control hiPSC-RPE stained for Phalloidin, and the RPE markers CRALBP, OTX2 and MCT1 (Scale bar: 100µM). Insets show digitally zoomed high magnification images. (C) AMD and control hiPSC-RPE showed similar TER. (D) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for AMD/drusen transcripts. (E-F) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of Aβ peptides (E) and VEGF-A (F). (G) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for complement/inflammatory transcripts. (H) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of C3. Data are expressed as mean± SEM. Unpaired Student’s t test (one-tailed) (D–H) and two-way ANOVA (D, G) were used for statistical analysis (*= p

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, One-tailed Test

    20) Product Images from "Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo"

    Article Title: Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo

    Journal: Scientific Reports

    doi: 10.1038/srep21961

    ( a ) A representative electron microscopic image of EVs derived from BMSCs (White arrow). Scale bar, 200 nm. The western blot of EV-depleted medium and EVs pallet also demonstrated that BMSC-derived EVs were isolated. For the FACS analysis, EVs (open trace) and negative control (filled trace) are shown. ( b ) Confocal fluorescence analysis was performed 4 h after incubation. The EVs were PKH67-labeled according to the manufacturer’s protocol (green fluorescence). The endoplasmic reticulum, Golgi apparatus and lysosomes were stained by ER-Tracker Red, Golgi-RFP and Lyso-Tracker Red. DAPI was used to stain the cell nuclei (blue fluorescence). Red arrows showed shallow green round-like staining indicating that the foreign EVs were degraded. The white arrow showed the round Golgi apparatus lumen.
    Figure Legend Snippet: ( a ) A representative electron microscopic image of EVs derived from BMSCs (White arrow). Scale bar, 200 nm. The western blot of EV-depleted medium and EVs pallet also demonstrated that BMSC-derived EVs were isolated. For the FACS analysis, EVs (open trace) and negative control (filled trace) are shown. ( b ) Confocal fluorescence analysis was performed 4 h after incubation. The EVs were PKH67-labeled according to the manufacturer’s protocol (green fluorescence). The endoplasmic reticulum, Golgi apparatus and lysosomes were stained by ER-Tracker Red, Golgi-RFP and Lyso-Tracker Red. DAPI was used to stain the cell nuclei (blue fluorescence). Red arrows showed shallow green round-like staining indicating that the foreign EVs were degraded. The white arrow showed the round Golgi apparatus lumen.

    Techniques Used: Derivative Assay, Western Blot, Isolation, FACS, Negative Control, Fluorescence, Incubation, Labeling, Staining

    ( a ) The RNA sequencing of BMSC and EVs indicated that miR-196a, miR27a and miR-206 were highly enriched in EVs. ( b ) Alizarin Red staining of osteoblasts treated with miR-196a, miR-27a and miR-206 at 3 days. ( c ) The OD ratio of Alizarin Red staining indicated a statistically significant difference compare miR-196a Mimic-group (0.42 ± 0.01) to the miR-27a Mimic-group (0.36 ± 0.01, P = 0.001, P
    Figure Legend Snippet: ( a ) The RNA sequencing of BMSC and EVs indicated that miR-196a, miR27a and miR-206 were highly enriched in EVs. ( b ) Alizarin Red staining of osteoblasts treated with miR-196a, miR-27a and miR-206 at 3 days. ( c ) The OD ratio of Alizarin Red staining indicated a statistically significant difference compare miR-196a Mimic-group (0.42 ± 0.01) to the miR-27a Mimic-group (0.36 ± 0.01, P = 0.001, P

    Techniques Used: RNA Sequencing Assay, Staining

    21) Product Images from "Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo"

    Article Title: Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo

    Journal: Scientific Reports

    doi: 10.1038/srep21961

    ( a ) A representative electron microscopic image of EVs derived from BMSCs (White arrow). Scale bar, 200 nm. The western blot of EV-depleted medium and EVs pallet also demonstrated that BMSC-derived EVs were isolated. For the FACS analysis, EVs (open trace) and negative control (filled trace) are shown. ( b ) Confocal fluorescence analysis was performed 4 h after incubation. The EVs were PKH67-labeled according to the manufacturer’s protocol (green fluorescence). The endoplasmic reticulum, Golgi apparatus and lysosomes were stained by ER-Tracker Red, Golgi-RFP and Lyso-Tracker Red. DAPI was used to stain the cell nuclei (blue fluorescence). Red arrows showed shallow green round-like staining indicating that the foreign EVs were degraded. The white arrow showed the round Golgi apparatus lumen.
    Figure Legend Snippet: ( a ) A representative electron microscopic image of EVs derived from BMSCs (White arrow). Scale bar, 200 nm. The western blot of EV-depleted medium and EVs pallet also demonstrated that BMSC-derived EVs were isolated. For the FACS analysis, EVs (open trace) and negative control (filled trace) are shown. ( b ) Confocal fluorescence analysis was performed 4 h after incubation. The EVs were PKH67-labeled according to the manufacturer’s protocol (green fluorescence). The endoplasmic reticulum, Golgi apparatus and lysosomes were stained by ER-Tracker Red, Golgi-RFP and Lyso-Tracker Red. DAPI was used to stain the cell nuclei (blue fluorescence). Red arrows showed shallow green round-like staining indicating that the foreign EVs were degraded. The white arrow showed the round Golgi apparatus lumen.

    Techniques Used: Derivative Assay, Western Blot, Isolation, FACS, Negative Control, Fluorescence, Incubation, Labeling, Staining

    22) Product Images from "Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells"

    Article Title: Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells

    Journal: Iranian Journal of Basic Medical Sciences

    doi:

    RT-PCR analysis of cytokeratin 18 and 19 in hepG2, differentiated cell on polystyrene and laminin
    Figure Legend Snippet: RT-PCR analysis of cytokeratin 18 and 19 in hepG2, differentiated cell on polystyrene and laminin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Flowcytometric analysis of C-MET expression with WinMDI 2.8 software. C-MET expression in human bMSCs (day 0) (12.39%) (panel a), hepG2 (93.35%) (panel b), 10th day of differentiation in cells on polystyrene (3.5%) (panel c) and on laminin (3.8%) (panel d), and 21th day of differentiation in cells on polystyrene (34.6%) (panel e) and on laminin (36.20%) (panel f)
    Figure Legend Snippet: Flowcytometric analysis of C-MET expression with WinMDI 2.8 software. C-MET expression in human bMSCs (day 0) (12.39%) (panel a), hepG2 (93.35%) (panel b), 10th day of differentiation in cells on polystyrene (3.5%) (panel c) and on laminin (3.8%) (panel d), and 21th day of differentiation in cells on polystyrene (34.6%) (panel e) and on laminin (36.20%) (panel f)

    Techniques Used: Expressing, Software

    23) Product Images from "Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human"

    Article Title: Structural and Functional Differences in the Long Non-Coding RNAHotair in Mouse and Human

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002071

    ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.
    Figure Legend Snippet: ChIP and expression profiling of control and Hoxc −/− MEFs. Enrichment of tri-methylated H3K27 over the HoxD gene cluster in both control mice and mice carrying a deletion of the HoxC cluster. The presence of this histone modification is assayed by qPCR after chromatin immunoprecipitation, either from dissected fetal hindbody (A) or from fetal hindlimbs at E13.5 (B). (C) Quantification of Hoxd gene transcripts present in either control, or HoxC mutant mouse embryonic fibroblasts (MEFs). (D) Comparison of H3K27me3 coverage between control and HoxC mutant-derived MEFs.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Methylation, Mouse Assay, Modification, Real-time Polymerase Chain Reaction, Mutagenesis, Derivative Assay

    Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.
    Figure Legend Snippet: Expression analysis of different Hoxd genes in control and HoxC mutant mice. (A) Schematic representation of the wild type and the HoxC deleted allele. (B) Absolute and relative quantifications of posterior Hoxd genes transcripts and of mHotair in forebody, hindbody, forelimbs and hindlimbs of E13.5 embryos. All values are normalized to a housekeeping gene. Relative amounts were calculated as a ratio by forcing wild type values to 1. Accordingly, small values are over-represented, explaining why mHotair gives a signal after deletion of HoxC , even though it is obviously absent. (C) Whole mount in situ hybridization (WISH) of Hoxd10 on E12.5 developing embryos. The expression domains of Hoxd genes remain globally unchanged (D) Hoxd10 expression patterns in developing forelimbs and hindlimbs at three developmental stages. Expression domains of Hoxd genes remain globally unchanged at all stages of limb development examined.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, In Situ Hybridization

    RNA–seq profiles of control and HoxC mutant mice. RNA was extracted from the region enriched in mHotair transcripts at day 13.5, i.e. the posterior part of the fetus, including the tail, hindlimbs and the outgrowing genitalia. Plotted are mean values of 25 bp windows. (A) Transcription profiles of the four different Hox gene clusters. The positions of the genes are indicated below. (A) Expression profiles of all four Hox loci, shown with the orientation with respect to the centromers. The strong peak in the deleted HoxC cluster is a transcript induced over the second exon of Hoxc4 (non-deleted) after deletion of the cluster (see the text). (B) Examples of transcriptional variations induced by the deletion of the HoxC cluster, with some genes being slightly up-regulated ( Hoxa7 , Hoxb9 , Hoxd10 and Wsb1 ), some being down-regulated ( Igf2r , Slc15a2 , Asb4 ). Hoxd13 is shown as an unaffected control gene ( Hoxd13 ). (C) Percentage of genes either up-regulated or down-regulated in HoxC mutant animals, which were also reported to be the targets of SUZ12 in ES cells. The percentages are comparable, suggesting that capacity to recruit PRC2 may not be the main cause of the transcriptional variations observed in the HoxC mutant animals, in these tissues at this developmental time. (D) Absolute quantifications of posterior Hoxd gene transcripts and of mHotair in posterior parts of fetuses including the hindlimbs, the genital bud and the developing tail of E11.5 embryos. All values are normalized to a housekeeping gene.
    Figure Legend Snippet: RNA–seq profiles of control and HoxC mutant mice. RNA was extracted from the region enriched in mHotair transcripts at day 13.5, i.e. the posterior part of the fetus, including the tail, hindlimbs and the outgrowing genitalia. Plotted are mean values of 25 bp windows. (A) Transcription profiles of the four different Hox gene clusters. The positions of the genes are indicated below. (A) Expression profiles of all four Hox loci, shown with the orientation with respect to the centromers. The strong peak in the deleted HoxC cluster is a transcript induced over the second exon of Hoxc4 (non-deleted) after deletion of the cluster (see the text). (B) Examples of transcriptional variations induced by the deletion of the HoxC cluster, with some genes being slightly up-regulated ( Hoxa7 , Hoxb9 , Hoxd10 and Wsb1 ), some being down-regulated ( Igf2r , Slc15a2 , Asb4 ). Hoxd13 is shown as an unaffected control gene ( Hoxd13 ). (C) Percentage of genes either up-regulated or down-regulated in HoxC mutant animals, which were also reported to be the targets of SUZ12 in ES cells. The percentages are comparable, suggesting that capacity to recruit PRC2 may not be the main cause of the transcriptional variations observed in the HoxC mutant animals, in these tissues at this developmental time. (D) Absolute quantifications of posterior Hoxd gene transcripts and of mHotair in posterior parts of fetuses including the hindlimbs, the genital bud and the developing tail of E11.5 embryos. All values are normalized to a housekeeping gene.

    Techniques Used: RNA Sequencing Assay, Mutagenesis, Mouse Assay, Expressing

    24) Product Images from "A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture"

    Article Title: A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

    Journal: eLife

    doi: 10.7554/eLife.51276

    In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.
    Figure Legend Snippet: In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.

    Techniques Used: In Vivo, FACS, Purification, Mouse Assay, RNA Sequencing Assay, Produced

    25) Product Images from "A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture"

    Article Title: A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

    Journal: eLife

    doi: 10.7554/eLife.51276

    In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.
    Figure Legend Snippet: In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.

    Techniques Used: In Vivo, FACS, Purification, Mouse Assay, RNA Sequencing Assay, Produced

    26) Product Images from "Comparative transcriptome analysis reveals a regulatory network of microRNA-29b during mouse early embryonic development"

    Article Title: Comparative transcriptome analysis reveals a regulatory network of microRNA-29b during mouse early embryonic development

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10741

    Experimental strategy for comparative transcriptome analysis A. Schematic depiction of preimplantation development following microinjection of miR29b inhibitor or vehicle control. Embryos were collected at the morula stage; B. cDNA library build and sequencing; C. Workflow of standard bioinformatics analysis and validation.
    Figure Legend Snippet: Experimental strategy for comparative transcriptome analysis A. Schematic depiction of preimplantation development following microinjection of miR29b inhibitor or vehicle control. Embryos were collected at the morula stage; B. cDNA library build and sequencing; C. Workflow of standard bioinformatics analysis and validation.

    Techniques Used: cDNA Library Assay, Sequencing

    A. MicroRNA target analysis of UDEGs. Left and right Y-axes represent number of UDEGs and P-value of enrichment, respectively. B. Heat map cluster analysis of transcript levels of candidate genes in normal embryos at the 1-cell, 2-cell and 4-cell stage. Normalized FPKM values are represented from blue to yellow; C. Relative expression of 6 UDEGs in morula stage embryos that had been injected with miR-29b inhibitor (red) or mock control (blue) at the zygote stage. The 6 genes analyzed are normally upregulated during early preimplantation stages (marked by red box in B). ***, P-value
    Figure Legend Snippet: A. MicroRNA target analysis of UDEGs. Left and right Y-axes represent number of UDEGs and P-value of enrichment, respectively. B. Heat map cluster analysis of transcript levels of candidate genes in normal embryos at the 1-cell, 2-cell and 4-cell stage. Normalized FPKM values are represented from blue to yellow; C. Relative expression of 6 UDEGs in morula stage embryos that had been injected with miR-29b inhibitor (red) or mock control (blue) at the zygote stage. The 6 genes analyzed are normally upregulated during early preimplantation stages (marked by red box in B). ***, P-value

    Techniques Used: Expressing, Injection

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

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

    Journal: eLife

    doi: 10.7554/eLife.36187

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

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

    28) Product Images from "Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females"

    Article Title: Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092841

    Expression of MPF components and its activity is increased in the oocytes from aged females. ( a ) RT-PCR quantification of mRNA coding for CDK1 and B-type cyclins, as well as loading control Gapdh in the GV oocytes (0 h) from different age groups. For quantification of total RNA content in oocytes from YF and AF groups see Figure S3a . Values obtained for the YF group were set as 100%. Data was derived from at least four experiments of biologically different samples. Columns represent mean; error bars ± SD; ns non-significant; * p
    Figure Legend Snippet: Expression of MPF components and its activity is increased in the oocytes from aged females. ( a ) RT-PCR quantification of mRNA coding for CDK1 and B-type cyclins, as well as loading control Gapdh in the GV oocytes (0 h) from different age groups. For quantification of total RNA content in oocytes from YF and AF groups see Figure S3a . Values obtained for the YF group were set as 100%. Data was derived from at least four experiments of biologically different samples. Columns represent mean; error bars ± SD; ns non-significant; * p

    Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    29) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    30) Product Images from "Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine"

    Article Title: Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0212369

    TER values of hiPS-RPE cell sheets cultured by machine and manually. hiPS-RPE cell sheets were cultured manually or using the automated ACE3 system and analyzed 49 days after seeding. The TER values of the hiPS-RPE cell sheets were calculated by subtracting the value of inserts covered with collagen gels as a blank from those of the experimental inserts. Data were obtained from two independent experiments. Machine cell culture, n = 9, manual cell culture, n = 7. All data are represented as the means ± SD.
    Figure Legend Snippet: TER values of hiPS-RPE cell sheets cultured by machine and manually. hiPS-RPE cell sheets were cultured manually or using the automated ACE3 system and analyzed 49 days after seeding. The TER values of the hiPS-RPE cell sheets were calculated by subtracting the value of inserts covered with collagen gels as a blank from those of the experimental inserts. Data were obtained from two independent experiments. Machine cell culture, n = 9, manual cell culture, n = 7. All data are represented as the means ± SD.

    Techniques Used: Cell Culture

    Phase-contrast and fluorescence images of immunostaining. hiPS-RPE cell sheets were cultured for 49 days using both machine and manual culture methods. (A–E) Phase-contrast image (top of each figure) and corresponding fluorescence image (bottom of each figure) of vertical sections of machine-cultured hiPS-RPE cell sheets. (A) Immunofluorescence detection of Na/K ATPase, (B) MERTK, (C) Claudin19, (D) RPE65, and (E) PMEL17. (F–J) Phase-contrast image (top) and corresponding fluorescence image (bottom) of vertical sections of manually cultured hiPS-RPE cell sheets. (F) Immunofluorescence detection of Na, K ATPase, (G) MERTK, (H) Claudin19, (I) RPE65, and (J) PMEL17. Nuclei were stained with DAPI. Scale bars: 20 μm.
    Figure Legend Snippet: Phase-contrast and fluorescence images of immunostaining. hiPS-RPE cell sheets were cultured for 49 days using both machine and manual culture methods. (A–E) Phase-contrast image (top of each figure) and corresponding fluorescence image (bottom of each figure) of vertical sections of machine-cultured hiPS-RPE cell sheets. (A) Immunofluorescence detection of Na/K ATPase, (B) MERTK, (C) Claudin19, (D) RPE65, and (E) PMEL17. (F–J) Phase-contrast image (top) and corresponding fluorescence image (bottom) of vertical sections of manually cultured hiPS-RPE cell sheets. (F) Immunofluorescence detection of Na, K ATPase, (G) MERTK, (H) Claudin19, (I) RPE65, and (J) PMEL17. Nuclei were stained with DAPI. Scale bars: 20 μm.

    Techniques Used: Fluorescence, Immunostaining, Cell Culture, Immunofluorescence, Staining

    Real-time PCR analysis of RPE-related gene expression in hiPS-RPE cell sheets. Data were obtained from two independent experiments. Machine cell culture, n = 7, manual cell culture, n = 5. All data are represented as the means ± SD.
    Figure Legend Snippet: Real-time PCR analysis of RPE-related gene expression in hiPS-RPE cell sheets. Data were obtained from two independent experiments. Machine cell culture, n = 7, manual cell culture, n = 5. All data are represented as the means ± SD.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    Phase-contrast and fluorescence images of immunostaining. hiPS-RPE cell sheets were cultured for 49 days using both machine and manual culture methods. (A) Schematic figure showing a cross-section of the RPE cell sheet. (B–G) Phase-contrast (B, C) and fluorescence (D–G) images of machine-cultured hiPS-RPE cell sheets. (B, D, F) Immunostaining for tight junction (ZO-1, red) and basement membrane (laminin, green) proteins. (C, E, G) Immunostaining for tight junction (ZO-1, green) and basement membrane (type IV collagen, red) proteins. (H–M) Phase-contrast (H, I) and fluorescence (J–M) images of manually cultured hiPS-RPE cell sheets. (H, J, L) Immunostaining for tight junction (ZO-1, red) and basement membrane (laminin, green) proteins. (I, K, M) Immunostaining for tight junction (ZO-1, green) and basement membrane (type IV collagen, red) proteins. Scale bars: 50 μm.
    Figure Legend Snippet: Phase-contrast and fluorescence images of immunostaining. hiPS-RPE cell sheets were cultured for 49 days using both machine and manual culture methods. (A) Schematic figure showing a cross-section of the RPE cell sheet. (B–G) Phase-contrast (B, C) and fluorescence (D–G) images of machine-cultured hiPS-RPE cell sheets. (B, D, F) Immunostaining for tight junction (ZO-1, red) and basement membrane (laminin, green) proteins. (C, E, G) Immunostaining for tight junction (ZO-1, green) and basement membrane (type IV collagen, red) proteins. (H–M) Phase-contrast (H, I) and fluorescence (J–M) images of manually cultured hiPS-RPE cell sheets. (H, J, L) Immunostaining for tight junction (ZO-1, red) and basement membrane (laminin, green) proteins. (I, K, M) Immunostaining for tight junction (ZO-1, green) and basement membrane (type IV collagen, red) proteins. Scale bars: 50 μm.

    Techniques Used: Fluorescence, Immunostaining, Cell Culture

    31) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    32) Product Images from "Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity"

    Article Title: Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.122299

    Modulating ROS levels regulates effector and memory CD8 + T cell differentiation in vitro. ( A ) Representative FACS analysis of CCR7 and CD45RO expression in circulating CD8 + Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of N-acetylcysteine (NAC), reduced glutathione (GSH), vitamin C (vitC), or apocynin (Apo) for 8 days. Treatments were supplemented daily. Additional DMSO control for Apo is shown. Similar data were obtained from n = 8 HD in n = 4 experiments (exp.) (NAC) and n = 3 HD in n = 1 exp. (GSH, vitC, Apo). ( B ) Representative histogram of CFSE dilution of cells cultured, as in A . NS, CFSE-stained, nonproliferating control cells. ( C ) Fold change (mean ± SEM) in cell counts compared with baseline ( n = 11 HD, n = 6 exp.) and ( D ) MFI (mean ± SEM) of CellROX, MitoSOX, and TMRM ( n = 9 HD, n = 4 exp.) indicative of total ROS levels, O 2 •– , and ΔΨ m , respectively, in CTRL and NAC-treated Tn cells at day 8 of culture, treated as in A . NAC was replaced every 3 days. ( E ) Representative CCR7 and CD45RO expression, as detected by FACS, of Tn cells activated as in A . NAC and menadione (MD) were replaced every 3 days. ( F ) FACS analysis of CD45RA, CD27, CD95, and CXCR3 by cells cultured, as in E . PBMCs from a HD are depicted as additional staining control. ( G ) Proportion (mean ± SEM) of CD8 + T cells with the Tscm, Tcm, Tem, and Tte phenotypes (gated as in Methods) after culture in the indicated conditions (CTRL, n = 13; NAC, n = 13, MD, n = 5 from n = 7 [NAC] and n = 2 [MD] exp.). ( H ) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in G (CTRL and NAC, n = 18; MD, n = 5 from n = 9 [NAC] and n = 2 [MD] exp.). ( I ) Pies depicting combinations of cytokine production obtained after stimulation, as in H . In all figures showing FACS dot plots, numbers indicate the percentage of cells identified by the gate. Statistical analyses were performed with nonparametric paired Wilcoxon ( C , D , G , and H ) and permutation ( I ) tests. * P
    Figure Legend Snippet: Modulating ROS levels regulates effector and memory CD8 + T cell differentiation in vitro. ( A ) Representative FACS analysis of CCR7 and CD45RO expression in circulating CD8 + Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of N-acetylcysteine (NAC), reduced glutathione (GSH), vitamin C (vitC), or apocynin (Apo) for 8 days. Treatments were supplemented daily. Additional DMSO control for Apo is shown. Similar data were obtained from n = 8 HD in n = 4 experiments (exp.) (NAC) and n = 3 HD in n = 1 exp. (GSH, vitC, Apo). ( B ) Representative histogram of CFSE dilution of cells cultured, as in A . NS, CFSE-stained, nonproliferating control cells. ( C ) Fold change (mean ± SEM) in cell counts compared with baseline ( n = 11 HD, n = 6 exp.) and ( D ) MFI (mean ± SEM) of CellROX, MitoSOX, and TMRM ( n = 9 HD, n = 4 exp.) indicative of total ROS levels, O 2 •– , and ΔΨ m , respectively, in CTRL and NAC-treated Tn cells at day 8 of culture, treated as in A . NAC was replaced every 3 days. ( E ) Representative CCR7 and CD45RO expression, as detected by FACS, of Tn cells activated as in A . NAC and menadione (MD) were replaced every 3 days. ( F ) FACS analysis of CD45RA, CD27, CD95, and CXCR3 by cells cultured, as in E . PBMCs from a HD are depicted as additional staining control. ( G ) Proportion (mean ± SEM) of CD8 + T cells with the Tscm, Tcm, Tem, and Tte phenotypes (gated as in Methods) after culture in the indicated conditions (CTRL, n = 13; NAC, n = 13, MD, n = 5 from n = 7 [NAC] and n = 2 [MD] exp.). ( H ) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in G (CTRL and NAC, n = 18; MD, n = 5 from n = 9 [NAC] and n = 2 [MD] exp.). ( I ) Pies depicting combinations of cytokine production obtained after stimulation, as in H . In all figures showing FACS dot plots, numbers indicate the percentage of cells identified by the gate. Statistical analyses were performed with nonparametric paired Wilcoxon ( C , D , G , and H ) and permutation ( I ) tests. * P

    Techniques Used: Cell Differentiation, In Vitro, FACS, Expressing, Cell Culture, Staining, Transmission Electron Microscopy

    Early differentiated human CD8 + T cells display a substantial antioxidant phenotype. ( A ) Gene set enrichment analysis (GSEA) of glutathione-derived metabolic process signature (gene ontology c5.pb.v6.1) in Tn versus Tem and Tscm versus Tem CD8 + T cells. Net enrichment score (NES) values are shown. ( B ) Relative gene expression level of transcripts involved in the antioxidant response in Tn, Tscm, Tcm, and Tem CD8 + T cell subsets from n = 3 HD. ( C ) Representative FACS analysis of GSH levels (mBCI staining) in gated CD8 + T cell subsets from the peripheral blood of a healthy individual. ( D ) Mean ± SEM of mean fluorescence intensity (MFI) data, obtained as in C ( n = 10). HD, healthy donor; mBCI, monochlorobimane; FMO, fluorescence minus one control. In D statistical analysis was performed with parametric 1-way ANOVA test with Bonferroni post test. ** P
    Figure Legend Snippet: Early differentiated human CD8 + T cells display a substantial antioxidant phenotype. ( A ) Gene set enrichment analysis (GSEA) of glutathione-derived metabolic process signature (gene ontology c5.pb.v6.1) in Tn versus Tem and Tscm versus Tem CD8 + T cells. Net enrichment score (NES) values are shown. ( B ) Relative gene expression level of transcripts involved in the antioxidant response in Tn, Tscm, Tcm, and Tem CD8 + T cell subsets from n = 3 HD. ( C ) Representative FACS analysis of GSH levels (mBCI staining) in gated CD8 + T cell subsets from the peripheral blood of a healthy individual. ( D ) Mean ± SEM of mean fluorescence intensity (MFI) data, obtained as in C ( n = 10). HD, healthy donor; mBCI, monochlorobimane; FMO, fluorescence minus one control. In D statistical analysis was performed with parametric 1-way ANOVA test with Bonferroni post test. ** P

    Techniques Used: Derivative Assay, Transmission Electron Microscopy, Expressing, FACS, Staining, Fluorescence

    33) Product Images from "Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions"

    Article Title: Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0900-16.2016

    Gene expression profiling by RNAseq of RGCs acutely purified from wild-type and NPC1-deficient mice. A , Left, false-color micrographs of RGCs acutely purified from 1-week-old mice by immunopanning and subjected to nuclear staining with DAPI (blue) and to immunocytochemical staining for Thy1 (green). Scale bar, 20 μm. Right, percentage of Thy1+ cells after isolation from retinae of 1-week-old ( n = 9) and 2-week-old mice ( n = 5; t test). Immunopanning delivered ∼73,000 ± 8,000 Thy1-positive cells per 1-week-old mouse ( n = 5). B , Quantity of total RNA in purified RGCs per mouse ( n = 4 mice per genotype corresponding to biological replicates; t test). C , Heat map of Pearson's correlation coefficient showing the reproducibility of transcript counts among biological replicates. D , First factorial plane resulting from a correspondence analysis of variance-stabilized data with the x -axis and y -axis explaining 23 and 22% of the variability of the whole dataset, respectively. E , Mean fold changes of transcript counts in RGCs from 1-week-old mutant mice compared to wild-type littermates plotted against normalized mean counts of each transcript as revealed by RNAseq. Red dots indicate genes with an adjusted p value of
    Figure Legend Snippet: Gene expression profiling by RNAseq of RGCs acutely purified from wild-type and NPC1-deficient mice. A , Left, false-color micrographs of RGCs acutely purified from 1-week-old mice by immunopanning and subjected to nuclear staining with DAPI (blue) and to immunocytochemical staining for Thy1 (green). Scale bar, 20 μm. Right, percentage of Thy1+ cells after isolation from retinae of 1-week-old ( n = 9) and 2-week-old mice ( n = 5; t test). Immunopanning delivered ∼73,000 ± 8,000 Thy1-positive cells per 1-week-old mouse ( n = 5). B , Quantity of total RNA in purified RGCs per mouse ( n = 4 mice per genotype corresponding to biological replicates; t test). C , Heat map of Pearson's correlation coefficient showing the reproducibility of transcript counts among biological replicates. D , First factorial plane resulting from a correspondence analysis of variance-stabilized data with the x -axis and y -axis explaining 23 and 22% of the variability of the whole dataset, respectively. E , Mean fold changes of transcript counts in RGCs from 1-week-old mutant mice compared to wild-type littermates plotted against normalized mean counts of each transcript as revealed by RNAseq. Red dots indicate genes with an adjusted p value of

    Techniques Used: Expressing, Purification, Mouse Assay, Staining, Isolation, Mutagenesis

    34) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    35) Product Images from "Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes"

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5608-2

    Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM
    Figure Legend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Techniques Used: Lysis, Isolation

    Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit
    Figure Legend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Techniques Used: Lysis, Isolation, FACS

    Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P
    Figure Legend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Techniques Used: Purification, FACS, Quantitative RT-PCR, Isolation

    36) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    37) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    38) Product Images from "Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression"

    Article Title: Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17919

    Rationale behind the Apc-Smad4 double knockout (Apc cKO Smad4 cKO ) mouse model ( A ) Smad4 immuno-staining on prostate tissue from Apc flox and Apc cKO mice (scale bars = 100 μm); ( B ) Axin2 and Smad4 quantitative reverse-transcriptase PCR from Apc flox and Apc cKO prostate tissue (error bars represent standard deviation based on technical triplicates; *** P value
    Figure Legend Snippet: Rationale behind the Apc-Smad4 double knockout (Apc cKO Smad4 cKO ) mouse model ( A ) Smad4 immuno-staining on prostate tissue from Apc flox and Apc cKO mice (scale bars = 100 μm); ( B ) Axin2 and Smad4 quantitative reverse-transcriptase PCR from Apc flox and Apc cKO prostate tissue (error bars represent standard deviation based on technical triplicates; *** P value

    Techniques Used: Double Knockout, Immunostaining, Mouse Assay, Polymerase Chain Reaction, Standard Deviation

    39) Product Images from "Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging"

    Article Title: Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00065

    Expression of inflammatory, phagocytic and metabolism genes in LFD and HFD microglia. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS. Microglia were FACS isolated and RNA was extracted and quantified using RT-qPCR. RNA expression levels were normalized to Hmbs levels as in internal control and the expression levels in PBS-injected LFD mice were set at 1. Gene expression levels were compared between both PBS- and LPS-injected mice and between HFD and LFD animals. (A) proinflammatory cytokines ( Il-1 β, Il-6 , and Tnf -α), (B) immune response ( Spp1 and Cybb ), (C) phagocytosis ( Axl, Lgals3 ), and (D) metabolism ( Apoe ) genes were significantly upregulated after LPS injection, but no significant difference between HFD+LPS and LFD+LPS samples was detected. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p
    Figure Legend Snippet: Expression of inflammatory, phagocytic and metabolism genes in LFD and HFD microglia. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS. Microglia were FACS isolated and RNA was extracted and quantified using RT-qPCR. RNA expression levels were normalized to Hmbs levels as in internal control and the expression levels in PBS-injected LFD mice were set at 1. Gene expression levels were compared between both PBS- and LPS-injected mice and between HFD and LFD animals. (A) proinflammatory cytokines ( Il-1 β, Il-6 , and Tnf -α), (B) immune response ( Spp1 and Cybb ), (C) phagocytosis ( Axl, Lgals3 ), and (D) metabolism ( Apoe ) genes were significantly upregulated after LPS injection, but no significant difference between HFD+LPS and LFD+LPS samples was detected. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p

    Techniques Used: Expressing, Injection, FACS, Isolation, Quantitative RT-PCR, RNA Expression, Mouse Assay

    Expression of immune-related genes in the hypothalamus of PBS/LPS-treated LFD and HFD mice. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS, total RNA was extracted from the hypothalamus and analyzed using RT-qPCR. The gene expression of (A) proinflammatory cytokines ( Il-1β and Tnf-α ), (B) genes relating to immune response ( CD44, Cryab, Sirpa, Spp1, Ifitm3 , and Ifitm2 ), (C) phagocytic markers ( Axl, Lgals3, CD36 , and Clec7a ), and (D) genes relating to lipid metabolism ( Apoe, Csf-1, Lpl , and Lrp12 ) were compared between HFD and LFD animals, but also between samples with or without LPS injection. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p
    Figure Legend Snippet: Expression of immune-related genes in the hypothalamus of PBS/LPS-treated LFD and HFD mice. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS, total RNA was extracted from the hypothalamus and analyzed using RT-qPCR. The gene expression of (A) proinflammatory cytokines ( Il-1β and Tnf-α ), (B) genes relating to immune response ( CD44, Cryab, Sirpa, Spp1, Ifitm3 , and Ifitm2 ), (C) phagocytic markers ( Axl, Lgals3, CD36 , and Clec7a ), and (D) genes relating to lipid metabolism ( Apoe, Csf-1, Lpl , and Lrp12 ) were compared between HFD and LFD animals, but also between samples with or without LPS injection. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p

    Techniques Used: Expressing, Mouse Assay, Injection, Quantitative RT-PCR

    40) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    NAM suppresses production of AMD-related disease markers in AMD and control hiPSC-RPE (A) Percentage mRNA expression for AMD/drusen transcripts analyzed by qPCR in AMD and control hiPSC-RPE treated with 10mM NAM relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM. , lower panel). (C-F) Secretion of APOJ (C), VEGF-A (D), Aβ-40 (E) and Aβ-42 (F) measured by ELISA into the culture supernatant 60–72 hours after the last medium change in AMD and control hiPSC-RPE treated with 10mM NAM or vehicle. ; each sample is color matched across NAM and vehicle treatment. Paired Student’s t test (one-tailed) was used for statistical analysis (*= p
    Figure Legend Snippet: NAM suppresses production of AMD-related disease markers in AMD and control hiPSC-RPE (A) Percentage mRNA expression for AMD/drusen transcripts analyzed by qPCR in AMD and control hiPSC-RPE treated with 10mM NAM relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM. , lower panel). (C-F) Secretion of APOJ (C), VEGF-A (D), Aβ-40 (E) and Aβ-42 (F) measured by ELISA into the culture supernatant 60–72 hours after the last medium change in AMD and control hiPSC-RPE treated with 10mM NAM or vehicle. ; each sample is color matched across NAM and vehicle treatment. Paired Student’s t test (one-tailed) was used for statistical analysis (*= p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    hiPSC-RPE from total AMD and AMD ARMS2/HTRA1 donors express higher levels of disease-related markers compared to controls (A) Experiment schematic. (B) AMD and control hiPSC-RPE stained for Phalloidin, and the RPE markers CRALBP, OTX2 and MCT1 (Scale bar: 100µM). Insets show digitally zoomed high magnification images. (C) AMD and control hiPSC-RPE showed similar TER. (D) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for AMD/drusen transcripts. (E-F) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of Aβ peptides (E) and VEGF-A (F). (G) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for complement/inflammatory transcripts. (H) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of C3. Data are expressed as mean± SEM. Unpaired Student’s t test (one-tailed) (D–H) and two-way ANOVA (D, G) were used for statistical analysis (*= p
    Figure Legend Snippet: hiPSC-RPE from total AMD and AMD ARMS2/HTRA1 donors express higher levels of disease-related markers compared to controls (A) Experiment schematic. (B) AMD and control hiPSC-RPE stained for Phalloidin, and the RPE markers CRALBP, OTX2 and MCT1 (Scale bar: 100µM). Insets show digitally zoomed high magnification images. (C) AMD and control hiPSC-RPE showed similar TER. (D) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for AMD/drusen transcripts. (E-F) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of Aβ peptides (E) and VEGF-A (F). (G) qPCR analysis of AMD ARMS2/HTRA1 hiPSC-RPE and total AMD hiPSC-RPE versus control hiPSC-RPE for complement/inflammatory transcripts. (H) ELISA testing of culture supernatant from AMD ARMS2/HTRA1, total AMD and control hiPSC-RPE obtained 60–72 hours after the last medium change for secretion of C3. Data are expressed as mean± SEM. Unpaired Student’s t test (one-tailed) (D–H) and two-way ANOVA (D, G) were used for statistical analysis (*= p

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, One-tailed Test

    41) Product Images from "Targeting JNK pathway promotes human hematopoietic stem cell expansion"

    Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0072-8

    The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Flow Cytometry, Cell Counting

    JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Transplantation Assay, Injection, Two Tailed Test, Software

    JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p
    Figure Legend Snippet: JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p

    Techniques Used:

    JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p
    Figure Legend Snippet: JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p

    Techniques Used: In Vitro, Cell Culture

    JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p
    Figure Legend Snippet: JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Inhibition, Flow Cytometry, Cytometry, Transduction, shRNA, Transfection

    42) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    43) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    44) Product Images from "Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1"

    Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

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

    doi: 10.1158/1078-0432.CCR-17-1952

    Jagged1 is required for lapatinib-mediated increase in MFE and high expressing cells are tumor initiating with higher stem cell frequency A. HCC1954 cells were transfected with a scrambled control siRNA or two distinct Jagged1 siRNAs for 48 hours using RNAiMax. The cells were then treated with DMSO or lapatinib for 2 followed by re-transfection and then treatment was continued for 2 additional days. Post treatment, cells were plated and %MFE assessed after 7 days. B. Cells were harvested post-transfection and assessed for Jagged1 protein expression by Western blotting. Actin protein was detected as a loading control. C. Vehicle treated Jagged1-low cells and lapatinib treated Jagged1-high cells were sorted, as described previously. 10,000 sorted cells were resuspended into a Matrigel solution (1:1 Matrigel:PBS) and were then injected into mammary fat pads of 5 female athymic nude mice per group, and tumor incidence was assessed for up to 10 weeks. Based on the tumor incidence, a Kaplan-Meier curve was plotted and statistics were performed using the Log-rank (Mantel-Cox) test. D. and E. Vehicle treated Jagged1-low expressing and lapatinib treated Jagged1-high expressing cells were sorted followed by 10,000, 1,000 or 100 cells resuspended into a Matrigel solution (1:1 Matrigel:PBS) and injected into mammary fat pad of 8-10 athymic nude mice per group. Tumor incidence was assessed for each dilution of cells for 8 weeks and CSC frequency was calculated using Extreme Limiting Dilution Analysis (ELDA) software ( F ). Panels D and E show the presentative images of tumors and mice from the groups that were injected with 10,000 cells. Panel F shows the CSC frequency estimates and P values calculated using the ELDA software.
    Figure Legend Snippet: Jagged1 is required for lapatinib-mediated increase in MFE and high expressing cells are tumor initiating with higher stem cell frequency A. HCC1954 cells were transfected with a scrambled control siRNA or two distinct Jagged1 siRNAs for 48 hours using RNAiMax. The cells were then treated with DMSO or lapatinib for 2 followed by re-transfection and then treatment was continued for 2 additional days. Post treatment, cells were plated and %MFE assessed after 7 days. B. Cells were harvested post-transfection and assessed for Jagged1 protein expression by Western blotting. Actin protein was detected as a loading control. C. Vehicle treated Jagged1-low cells and lapatinib treated Jagged1-high cells were sorted, as described previously. 10,000 sorted cells were resuspended into a Matrigel solution (1:1 Matrigel:PBS) and were then injected into mammary fat pads of 5 female athymic nude mice per group, and tumor incidence was assessed for up to 10 weeks. Based on the tumor incidence, a Kaplan-Meier curve was plotted and statistics were performed using the Log-rank (Mantel-Cox) test. D. and E. Vehicle treated Jagged1-low expressing and lapatinib treated Jagged1-high expressing cells were sorted followed by 10,000, 1,000 or 100 cells resuspended into a Matrigel solution (1:1 Matrigel:PBS) and injected into mammary fat pad of 8-10 athymic nude mice per group. Tumor incidence was assessed for each dilution of cells for 8 weeks and CSC frequency was calculated using Extreme Limiting Dilution Analysis (ELDA) software ( F ). Panels D and E show the presentative images of tumors and mice from the groups that were injected with 10,000 cells. Panel F shows the CSC frequency estimates and P values calculated using the ELDA software.

    Techniques Used: Expressing, Transfection, Western Blot, Injection, Mouse Assay, Software

    Lapatinib induces Jagged1-high expressing cells with mammosphere forming potential HER2+ HCC1954 ( A ), MCF-7-HER2 ( B ), MDA-MB-453 ( C ), and HER2 wild type expressing MCF-7 ( D ) cells were treated for four days with 2 μM Lapatinib or vehicle (DMSO). Cells were harvested and stained for Jagged1 followed by flow cytometry. E. HCC1954 cells were stained for Jagged1 and then sorted by flow cytometry on the basis of Jagged1 surface expression. The Jagged1 -/low or high population was sorted from both vehicle and lapatinib treated cells. The schematic shows the gating for the cells that were sorted. 35,000 HCC1954 cells were sorted into a well of a 24 well ultra-low attachment plate containing mammosphere forming medium. After 7 days, the mammospheres were harvested and %MFE was determined. Scale bars = 100 μm.
    Figure Legend Snippet: Lapatinib induces Jagged1-high expressing cells with mammosphere forming potential HER2+ HCC1954 ( A ), MCF-7-HER2 ( B ), MDA-MB-453 ( C ), and HER2 wild type expressing MCF-7 ( D ) cells were treated for four days with 2 μM Lapatinib or vehicle (DMSO). Cells were harvested and stained for Jagged1 followed by flow cytometry. E. HCC1954 cells were stained for Jagged1 and then sorted by flow cytometry on the basis of Jagged1 surface expression. The Jagged1 -/low or high population was sorted from both vehicle and lapatinib treated cells. The schematic shows the gating for the cells that were sorted. 35,000 HCC1954 cells were sorted into a well of a 24 well ultra-low attachment plate containing mammosphere forming medium. After 7 days, the mammospheres were harvested and %MFE was determined. Scale bars = 100 μm.

    Techniques Used: Expressing, Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry

    Survival of lapatinib-induced Jagged1-high expressing CSCs is dependent on the γ-secretase complex HCC1954 ( A ) or MCF-7-HER2 ( B ) cells were treated with DMSO (Vehicle) or lapatinib for 4 days followed by flow cytometry to detect cell surface expression of Jagged1. Cells were sorted for low versus high Jagged1 expressing cells and plated onto low attachment plates-containing mammosphere forming medium-supplemented with DMSO (Control) or 5μM MRK-003 GSI. %MFE was assessed at day 7. Scale bar = 100μm. *Denotes statistical significance of P
    Figure Legend Snippet: Survival of lapatinib-induced Jagged1-high expressing CSCs is dependent on the γ-secretase complex HCC1954 ( A ) or MCF-7-HER2 ( B ) cells were treated with DMSO (Vehicle) or lapatinib for 4 days followed by flow cytometry to detect cell surface expression of Jagged1. Cells were sorted for low versus high Jagged1 expressing cells and plated onto low attachment plates-containing mammosphere forming medium-supplemented with DMSO (Control) or 5μM MRK-003 GSI. %MFE was assessed at day 7. Scale bar = 100μm. *Denotes statistical significance of P

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Lapatinib-induced Jagged1-high cells express higher Notch receptors and gene targets HCC1954 vehicle treated Jagged1-low cells and lapatinib treated Jagged1-high cells at a density of 400,000 cells were sorted into a collection tube. After the sort, RNA was extracted from sorted cells followed by cDNA synthesis using reverse transcription and real-time PCR to detect transcript levels of Notch receptors ( A ) and target genes ( B ) in the two populations. Bar graphs show mean values of relative transcript expression normalized to HPRT and compared to Vehicle Jagged1-low ± S.D. from three independent experiments using the 2 −ΔΔCt calculation. *, P
    Figure Legend Snippet: Lapatinib-induced Jagged1-high cells express higher Notch receptors and gene targets HCC1954 vehicle treated Jagged1-low cells and lapatinib treated Jagged1-high cells at a density of 400,000 cells were sorted into a collection tube. After the sort, RNA was extracted from sorted cells followed by cDNA synthesis using reverse transcription and real-time PCR to detect transcript levels of Notch receptors ( A ) and target genes ( B ) in the two populations. Bar graphs show mean values of relative transcript expression normalized to HPRT and compared to Vehicle Jagged1-low ± S.D. from three independent experiments using the 2 −ΔΔCt calculation. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    45) Product Images from "Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells"

    Article Title: Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084072

    Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.
    Figure Legend Snippet: Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.

    Techniques Used: Expressing, Microarray

    46) Product Images from "Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo"

    Article Title: Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo

    Journal: Scientific Reports

    doi: 10.1038/srep21961

    ( a ) The RNA sequencing of BMSC and EVs indicated that miR-196a, miR27a and miR-206 were highly enriched in EVs. ( b ) Alizarin Red staining of osteoblasts treated with miR-196a, miR-27a and miR-206 at 3 days. ( c ) The OD ratio of Alizarin Red staining indicated a statistically significant difference compare miR-196a Mimic-group (0.42 ± 0.01) to the miR-27a Mimic-group (0.36 ± 0.01, P = 0.001, P
    Figure Legend Snippet: ( a ) The RNA sequencing of BMSC and EVs indicated that miR-196a, miR27a and miR-206 were highly enriched in EVs. ( b ) Alizarin Red staining of osteoblasts treated with miR-196a, miR-27a and miR-206 at 3 days. ( c ) The OD ratio of Alizarin Red staining indicated a statistically significant difference compare miR-196a Mimic-group (0.42 ± 0.01) to the miR-27a Mimic-group (0.36 ± 0.01, P = 0.001, P

    Techniques Used: RNA Sequencing Assay, Staining

    47) Product Images from "Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae"

    Article Title: Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.221169

    Adenovirus infection rate in the organ of Corti depends on width of the sensory epithelium. Representative images of myosin7A and Sox2 staining and immunofluorescence were observed using a Zeiss confocal microscope. (A) The organ of Corti was well organized when samples were fixed immediately after dissection. (B) Cochlear explants were cultured in medium containing 5 % FBS for 24 hours after neomycin insult and were then infected with Ad5-GFP . (C) Cochlear explants were cultured in medium containing 5 % FBS for 48 hours after neomycin insult and were then infected with Ad5-GFP . (D) Cochlear explants were cultured in medium containing 10 % FBS for 48 hours after neomycin insult and were then infected with Ad5-GFP . Many GFP-immunoreactive cells were observed in the sensory epithelium. (E) The bar graph shows the width of sensory epithelium. (F) The bar graph shows the infection rate of Ad5; * P
    Figure Legend Snippet: Adenovirus infection rate in the organ of Corti depends on width of the sensory epithelium. Representative images of myosin7A and Sox2 staining and immunofluorescence were observed using a Zeiss confocal microscope. (A) The organ of Corti was well organized when samples were fixed immediately after dissection. (B) Cochlear explants were cultured in medium containing 5 % FBS for 24 hours after neomycin insult and were then infected with Ad5-GFP . (C) Cochlear explants were cultured in medium containing 5 % FBS for 48 hours after neomycin insult and were then infected with Ad5-GFP . (D) Cochlear explants were cultured in medium containing 10 % FBS for 48 hours after neomycin insult and were then infected with Ad5-GFP . Many GFP-immunoreactive cells were observed in the sensory epithelium. (E) The bar graph shows the width of sensory epithelium. (F) The bar graph shows the infection rate of Ad5; * P

    Techniques Used: Infection, Staining, Immunofluorescence, Microscopy, Dissection, Cell Culture

    Atoh1 overexpression induced ectopic hair cell formation in the lesser epithelial ridge. Representative images of myosin7A staining and immunofluorescence were observed using a Zeiss confocal microscope. We cultured cochlear explants of neonatal rats in medium containing 5% fetal bovine serum for 24 hours then infected them with Ad5-GFP-Atoh1 . (A) Eight days after Atoh1 infection, GFP-Myosin 7A double immuno-reactive cells were observed to be restricted to the lesser epithelial ridge. (B) Magnified view of (A): White arrows point to the newly generated hair cells. (C) Eight days after infection with GFP alone, no myosin 7A-GFP double-immunoreactive cells could be detected in the lesser epithelial ridge. The virus vector failed to infect the sensory epithelium (white brackets) and no Myosin 7A-immunoreactive cells were found in situ . Green: GFP-immuno-reactive cells; blue: myosin7A-immunoreactive hair cells. Scale bars: 100 μm in A, C; 50 μm in B. Atoh1: Atonal homolog 1; Ad5: adenovirus type 5; GFP: green fluorescent protein.
    Figure Legend Snippet: Atoh1 overexpression induced ectopic hair cell formation in the lesser epithelial ridge. Representative images of myosin7A staining and immunofluorescence were observed using a Zeiss confocal microscope. We cultured cochlear explants of neonatal rats in medium containing 5% fetal bovine serum for 24 hours then infected them with Ad5-GFP-Atoh1 . (A) Eight days after Atoh1 infection, GFP-Myosin 7A double immuno-reactive cells were observed to be restricted to the lesser epithelial ridge. (B) Magnified view of (A): White arrows point to the newly generated hair cells. (C) Eight days after infection with GFP alone, no myosin 7A-GFP double-immunoreactive cells could be detected in the lesser epithelial ridge. The virus vector failed to infect the sensory epithelium (white brackets) and no Myosin 7A-immunoreactive cells were found in situ . Green: GFP-immuno-reactive cells; blue: myosin7A-immunoreactive hair cells. Scale bars: 100 μm in A, C; 50 μm in B. Atoh1: Atonal homolog 1; Ad5: adenovirus type 5; GFP: green fluorescent protein.

    Techniques Used: Over Expression, Staining, Immunofluorescence, Microscopy, Cell Culture, Infection, Generated, Plasmid Preparation, In Situ

    DAPT enhances Atoh1 activity to generate more new hair cells in situ . Representative images of myosin7A and Sox2 staining and immunofluorescence were observed using a Zeiss confocal microscope. Neomycin (1.5 mM) was applied to destroy almost all of the normal hair cells and Ad5-GFP or Ad5-GFP-Atoh1 was applied after 48 hours of culture in 10 % FBS medium. (A) Cochlear explants were infected by Ad5-GFP after neomycin insult. No myosin 7A-immunoreactive cells were found in the sensory epithelium. (B) Cochlear explants were treated with Ad5-GFP and DAPT . No myosin 7A-immunoreactive cells were detected in the sensory epithelium. (C) Cochlear explants were infected by Ad5-GFP-Atoh1 after neomycin insult. Several GFP/myosin 7A double immunoreactive cells were observed in the sensory epithelium. (D) Cochlear explants with concurrent application of Ad5-GFP-Atoh1 and DAPT after neomycin insult. Numerous GFP/myosin 7A double immunoreactive cells were found in the sensory epithelium. (A1–D1) Magnified view from A–D. (E) The number of new hair cells in situ in the different groups ( n = 10). (F) Quantitative polymerase chain reaction results for the different groups; * P
    Figure Legend Snippet: DAPT enhances Atoh1 activity to generate more new hair cells in situ . Representative images of myosin7A and Sox2 staining and immunofluorescence were observed using a Zeiss confocal microscope. Neomycin (1.5 mM) was applied to destroy almost all of the normal hair cells and Ad5-GFP or Ad5-GFP-Atoh1 was applied after 48 hours of culture in 10 % FBS medium. (A) Cochlear explants were infected by Ad5-GFP after neomycin insult. No myosin 7A-immunoreactive cells were found in the sensory epithelium. (B) Cochlear explants were treated with Ad5-GFP and DAPT . No myosin 7A-immunoreactive cells were detected in the sensory epithelium. (C) Cochlear explants were infected by Ad5-GFP-Atoh1 after neomycin insult. Several GFP/myosin 7A double immunoreactive cells were observed in the sensory epithelium. (D) Cochlear explants with concurrent application of Ad5-GFP-Atoh1 and DAPT after neomycin insult. Numerous GFP/myosin 7A double immunoreactive cells were found in the sensory epithelium. (A1–D1) Magnified view from A–D. (E) The number of new hair cells in situ in the different groups ( n = 10). (F) Quantitative polymerase chain reaction results for the different groups; * P

    Techniques Used: Activity Assay, In Situ, Staining, Immunofluorescence, Microscopy, Infection, Real-time Polymerase Chain Reaction

    48) Product Images from "CD177-mediated nanoparticle targeting of human and mouse neutrophils"

    Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200444

    Mouse C5aR1 ASO results in knockdown of mouse C5aR1-GFP in CHO transfectants. CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value
    Figure Legend Snippet: Mouse C5aR1 ASO results in knockdown of mouse C5aR1-GFP in CHO transfectants. CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value

    Techniques Used: Allele-specific Oligonucleotide, Expressing, Transfection, Flow Cytometry, Cytometry, Negative Control, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Mouse C5aR1 siRNA and human C5aR1 siRNA pool result in receptor knockdown. CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.
    Figure Legend Snippet: Mouse C5aR1 siRNA and human C5aR1 siRNA pool result in receptor knockdown. CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.

    Techniques Used: Expressing, Transfection, Positive Control, Negative Control, Flow Cytometry, Cytometry

    49) Product Images from "Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis"

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    Journal: eLife

    doi: 10.7554/eLife.40556

    let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).
    Figure Legend Snippet: let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).

    Techniques Used: Binding Assay, Reporter Assay, Sequencing, Transfection, Construct, Fluorescence, Immunostaining, Expressing, Staining

    The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.
    Figure Legend Snippet: The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.

    Techniques Used: In Vivo, Binding Assay, Activity Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Injection, Immunohistochemistry, Expressing, Plasmid Preparation, Construct, Quantitation Assay

    50) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    51) Product Images from "Targeting JNK pathway promotes human hematopoietic stem cell expansion"

    Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0072-8

    The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Flow Cytometry, Cell Counting

    JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Transplantation Assay, Injection, Two Tailed Test, Software

    JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p
    Figure Legend Snippet: JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p

    Techniques Used:

    JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p
    Figure Legend Snippet: JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p

    Techniques Used: In Vitro, Cell Culture

    JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p
    Figure Legend Snippet: JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Inhibition, Flow Cytometry, Cytometry, Transduction, shRNA, Transfection

    52) Product Images from "Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine"

    Article Title: Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0212369

    Real-time PCR analysis of RPE-related gene expression in hiPS-RPE cell sheets. Data were obtained from two independent experiments. Machine cell culture, n = 7, manual cell culture, n = 5. All data are represented as the means ± SD.
    Figure Legend Snippet: Real-time PCR analysis of RPE-related gene expression in hiPS-RPE cell sheets. Data were obtained from two independent experiments. Machine cell culture, n = 7, manual cell culture, n = 5. All data are represented as the means ± SD.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    53) Product Images from "Targeting JNK pathway promotes human hematopoietic stem cell expansion"

    Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0072-8

    The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Flow Cytometry, Cell Counting

    JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Transplantation Assay, Injection, Two Tailed Test, Software

    JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p
    Figure Legend Snippet: JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p

    Techniques Used:

    JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1 . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p
    Figure Legend Snippet: JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1 . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p

    Techniques Used: In Vitro, Cell Culture, Concentration Assay

    JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p
    Figure Legend Snippet: JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Inhibition, Flow Cytometry, Cytometry, Transduction, shRNA, Transfection

    54) Product Images from "Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition"

    Article Title: Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition

    Journal: Nature Communications

    doi: 10.1038/ncomms8739

    Endothelial to haematopoietic conversion is increased after Sox17 loss. ( a ) Schema and bar graph of qRT–PCR analyses of sorted endothelial cells from E11 embryos after in vivo Sox17 ablation at E9.5. Error bars indicate standard error of the mean ( n =3 litters, embryos pooled by genotype). LOF, loss of function. ( b ) Immunofluorescence of Sox17 heterozygous and homozygous embryos at E10.5 after in vivo Cre induction (tamoxifen induction at E9.5). Haematopoietic clusters are labelled by CD117 (green), Cre traced endothelial and cluster cells in red (Td + ). SOX17 (grey) is absent in homozygous mutant endothelium. DAPI in blue. DA, dorsal aorta. Scale bar, 10 μm. Single channels in black and white. ( c ) Schematic of AGM explant analysis depicts in vitro Cre lineage tracing and calculation of hemogenic output (HE ratio); the ratio between per cent labelled (Td + ) haematopoietic cells (CD45 + CD31 − ) to per cent labelled (Td + ) endothelial cells (CD31 + CD45 − ). 4OHT, 4-hydroxytamoxifen. ( d – f ) Each data point represents a separate embryo/AGM explant, littermates are depicted by the same data point colour and shape. Bar indicates group mean. P values calculated on Student's t -test between groups, significance also validated by two-way analysis of variance, ( Supplementary Table 2 ). ( d ) The HE ratio of Sox17 homozygous (f/f) and heterozygous (f/+) mutant explants. f/+ n =45, f/f n =38, 15 litters. ( e ) Percentage of traced Td + hemogenic endothelial cluster cells, designated as CD31 + CD41 + . f/+ n =37, f/f n =26, 9 litters. ( f ) Percentage of traced (Td + ) maturing HSPCs (identified as CD31 + CD117 + Sca1 + CD45 + ), f/+ n =14, f/f n =27, 7 litters. ( g ) Schema depicts overexpression analyses in wild-type AGM explants at E11.0. ( h ) Immunofluorescence of E11 AGM explant after human adenoviral SOX17-GFP infection. GFP in green, SOX17 in magenta and DAPI in blue. Scale bar as indicated. ( i ) Cell sorting strategy for endothelial cells (CD31 + ) after exposure to AdhSOX17-GFP (GFP), where GFP + and GFP − populations were gated. ( j ) Bar graph of qRT–PCR analyses of sorted E11 AGM CD31 + cells after AdhSOX17-GFP infection. Error bars indicate s.e.m. CD31 + GFP − population served as a control, set to one for comparisons of fold change, n =3 litters, embryos pooled, P values as indicated. ( a , j ) P values reflect Student's t -test.
    Figure Legend Snippet: Endothelial to haematopoietic conversion is increased after Sox17 loss. ( a ) Schema and bar graph of qRT–PCR analyses of sorted endothelial cells from E11 embryos after in vivo Sox17 ablation at E9.5. Error bars indicate standard error of the mean ( n =3 litters, embryos pooled by genotype). LOF, loss of function. ( b ) Immunofluorescence of Sox17 heterozygous and homozygous embryos at E10.5 after in vivo Cre induction (tamoxifen induction at E9.5). Haematopoietic clusters are labelled by CD117 (green), Cre traced endothelial and cluster cells in red (Td + ). SOX17 (grey) is absent in homozygous mutant endothelium. DAPI in blue. DA, dorsal aorta. Scale bar, 10 μm. Single channels in black and white. ( c ) Schematic of AGM explant analysis depicts in vitro Cre lineage tracing and calculation of hemogenic output (HE ratio); the ratio between per cent labelled (Td + ) haematopoietic cells (CD45 + CD31 − ) to per cent labelled (Td + ) endothelial cells (CD31 + CD45 − ). 4OHT, 4-hydroxytamoxifen. ( d – f ) Each data point represents a separate embryo/AGM explant, littermates are depicted by the same data point colour and shape. Bar indicates group mean. P values calculated on Student's t -test between groups, significance also validated by two-way analysis of variance, ( Supplementary Table 2 ). ( d ) The HE ratio of Sox17 homozygous (f/f) and heterozygous (f/+) mutant explants. f/+ n =45, f/f n =38, 15 litters. ( e ) Percentage of traced Td + hemogenic endothelial cluster cells, designated as CD31 + CD41 + . f/+ n =37, f/f n =26, 9 litters. ( f ) Percentage of traced (Td + ) maturing HSPCs (identified as CD31 + CD117 + Sca1 + CD45 + ), f/+ n =14, f/f n =27, 7 litters. ( g ) Schema depicts overexpression analyses in wild-type AGM explants at E11.0. ( h ) Immunofluorescence of E11 AGM explant after human adenoviral SOX17-GFP infection. GFP in green, SOX17 in magenta and DAPI in blue. Scale bar as indicated. ( i ) Cell sorting strategy for endothelial cells (CD31 + ) after exposure to AdhSOX17-GFP (GFP), where GFP + and GFP − populations were gated. ( j ) Bar graph of qRT–PCR analyses of sorted E11 AGM CD31 + cells after AdhSOX17-GFP infection. Error bars indicate s.e.m. CD31 + GFP − population served as a control, set to one for comparisons of fold change, n =3 litters, embryos pooled, P values as indicated. ( a , j ) P values reflect Student's t -test.

    Techniques Used: Quantitative RT-PCR, In Vivo, Immunofluorescence, Mutagenesis, In Vitro, Over Expression, Infection, FACS

    Haematopoietic cell clusters downregulate arterial gene expression. ( a – e ) Single channels in black and white, scale bars as shown. E10.5 wild-type dorsal aorta (DA). ( a ) Haematopoietic cell clusters of the AGM at E10.5. The endothelial layer and attached haematopoietic cell clusters are CD31 + (red). RUNX1 (grey) is notable in cells comprising the haematopoietic cluster (arrowhead). SOX17 (green) expression is localized to the endothelial layer (arrow). DAPI in blue. ( b ) GATA2 (green) is notable in the haematopoietic cell cluster (arrowhead). CD31 (red) and DAPI (blue). ( c ) SOX17 (green) immunofluorescence is noted in the cell nuclei of the endothelial layer (arrowheads), as compared with the associated cell cluster. CD31 in red, and DAPI in blue. ( d ) Notch pathway activation (green) as measured in the TP1 Venus mouse line is notable in the endothelial layer (arrow) but less so in the associated haematopoietic cell cluster, CD31 in red. DAPI in blue. ( e ) CD144 (red) labels the endothelium and haematopoietic cluster cells (arrowhead), Sox17 in grey, and Runx1 in green. ( f ) Embryos at E10.5 were sorted based on cell surface markers to isolate endothelial cells (CD31 + CD117 − CD45 − ), haematopoietic cluster cells (CD31 + CD117 + CD45 − ), maturing cluster cells and HSPCs (CD31 + CD117 + CD45 + ) and mature haematopoietic cells (CD31 − CD45 + ). Bar graphs depict transcript expression (RT–PCR) in each subgroup for Runx1 , Gata2 , Sox17, Notch1 and Cdh5 (CD144). Differing letters represent significance between groups where a versus b, or b versus c, or a versus c, is significant to a P value
    Figure Legend Snippet: Haematopoietic cell clusters downregulate arterial gene expression. ( a – e ) Single channels in black and white, scale bars as shown. E10.5 wild-type dorsal aorta (DA). ( a ) Haematopoietic cell clusters of the AGM at E10.5. The endothelial layer and attached haematopoietic cell clusters are CD31 + (red). RUNX1 (grey) is notable in cells comprising the haematopoietic cluster (arrowhead). SOX17 (green) expression is localized to the endothelial layer (arrow). DAPI in blue. ( b ) GATA2 (green) is notable in the haematopoietic cell cluster (arrowhead). CD31 (red) and DAPI (blue). ( c ) SOX17 (green) immunofluorescence is noted in the cell nuclei of the endothelial layer (arrowheads), as compared with the associated cell cluster. CD31 in red, and DAPI in blue. ( d ) Notch pathway activation (green) as measured in the TP1 Venus mouse line is notable in the endothelial layer (arrow) but less so in the associated haematopoietic cell cluster, CD31 in red. DAPI in blue. ( e ) CD144 (red) labels the endothelium and haematopoietic cluster cells (arrowhead), Sox17 in grey, and Runx1 in green. ( f ) Embryos at E10.5 were sorted based on cell surface markers to isolate endothelial cells (CD31 + CD117 − CD45 − ), haematopoietic cluster cells (CD31 + CD117 + CD45 − ), maturing cluster cells and HSPCs (CD31 + CD117 + CD45 + ) and mature haematopoietic cells (CD31 − CD45 + ). Bar graphs depict transcript expression (RT–PCR) in each subgroup for Runx1 , Gata2 , Sox17, Notch1 and Cdh5 (CD144). Differing letters represent significance between groups where a versus b, or b versus c, or a versus c, is significant to a P value

    Techniques Used: Expressing, Immunofluorescence, Activation Assay, Reverse Transcription Polymerase Chain Reaction

    55) Product Images from "Lung CD103+ dendritic cells restrain allergic airway inflammation through IL-12 production"

    Article Title: Lung CD103+ dendritic cells restrain allergic airway inflammation through IL-12 production

    Journal: JCI Insight

    doi: 10.1172/jci.insight.90420

    CD103 + mig-DCs are the main DC source of IL-12 in mLNs after HDM exposure. Mice were challenged with 100 μg HDM i.n., and mLNs were collected 3 days later. ( A ) IL-12p40 and ( B ) IL-6 mRNA expression was analyzed in purified CD11c + cells, and mRNA was normalized against β-actin. Data shown (mean ± SEM) is a pool of 3 independent experiments; each symbol represents 1 experiment (5–10 mice pooled per experiment); ** P
    Figure Legend Snippet: CD103 + mig-DCs are the main DC source of IL-12 in mLNs after HDM exposure. Mice were challenged with 100 μg HDM i.n., and mLNs were collected 3 days later. ( A ) IL-12p40 and ( B ) IL-6 mRNA expression was analyzed in purified CD11c + cells, and mRNA was normalized against β-actin. Data shown (mean ± SEM) is a pool of 3 independent experiments; each symbol represents 1 experiment (5–10 mice pooled per experiment); ** P

    Techniques Used: Mouse Assay, Expressing, Purification

    56) Product Images from "Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes"

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5608-2

    Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM
    Figure Legend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Techniques Used: Lysis, Isolation

    Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit
    Figure Legend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Techniques Used: Lysis, Isolation, FACS

    Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P
    Figure Legend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Techniques Used: Purification, FACS, Quantitative RT-PCR, Isolation

    57) Product Images from "Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip"

    Article Title: Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.6b12236

    Characterization of the patient-derived breast (Brx) CTC line using imaging flow cytometry. Data compare viability, EpCAM expression, and area of control versus captured/released cells from our NP- HB CTC-Chip. Representative images of one viable, cluster, and dead Brx cell (A) obtained from culture (control) and (B) captured/released from our microfluidic device. Gate settings of (C) control and (D) captured/released Brx cells. Viable cells are defined as calcein positive and caspase 3/7 negative, whereas dead cells are caspase 3/7 positive. The intensity of EpCAM obtained from (E) control and (F) captured/released Brx cells. The area of (G) control and (H) captured/released Brx cells. (I) Heat map of the C t values of seven genes obtained by RT-qPCR. Comparisons were across control, released Brx cells, and white blood cells (WBC).
    Figure Legend Snippet: Characterization of the patient-derived breast (Brx) CTC line using imaging flow cytometry. Data compare viability, EpCAM expression, and area of control versus captured/released cells from our NP- HB CTC-Chip. Representative images of one viable, cluster, and dead Brx cell (A) obtained from culture (control) and (B) captured/released from our microfluidic device. Gate settings of (C) control and (D) captured/released Brx cells. Viable cells are defined as calcein positive and caspase 3/7 negative, whereas dead cells are caspase 3/7 positive. The intensity of EpCAM obtained from (E) control and (F) captured/released Brx cells. The area of (G) control and (H) captured/released Brx cells. (I) Heat map of the C t values of seven genes obtained by RT-qPCR. Comparisons were across control, released Brx cells, and white blood cells (WBC).

    Techniques Used: Derivative Assay, Imaging, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

    58) Product Images from "Impaired αVβ8 and TGFβ signaling lead to microglial dysmaturation and neuromotor dysfunction"

    Article Title: Impaired αVβ8 and TGFβ signaling lead to microglial dysmaturation and neuromotor dysfunction

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20181290

    Microglial dysmaturation due to loss of TGFβ signaling is critically time dependent. (A) Diagram of Tgfbr2 gene inactivation and mutant analysis. Tamoxifen was injected at P1 for early induction in B or at P30 for adult induction in C. Mice were sacrificed and analyzed at indicated time points after tamoxifen injection. (B and C) Staining of cortical brain sections from Tgfbr2iΔMG and Cre + control littermates 120 d after tamoxifen administration following early (B) or adult (C) induction. Left panels: tdTomato recombination reporter (tdTom, red) marks all microglia and immature (APOE, green) and mature (P2RY12, blue) microglia markers to identify type A (dysmature) or type B microglia, respectively. Right panels (insets from Fig. S5): pSMAD3 (green) coimmunostaining reveals reduction in pSMAD3 staining intensity in type A (dysmature) microglia compared with type B microglia and controls. Percent (%) recombination (upper right graphs in B and C) based on % F4/80 + , CD45 + , CD11b + cells that are also tdTomato positive (cells isolated and analyzed by flow cytometry as in Fig. S3). Mean pSMAD3 per nucleus (lower right graphs in B and C) based on fluorescent intensity of individual recombined (tdTom + ) microglia coexpressing P2RY12 (blue, control and type B cells), or lacking P2RY12 expression (type A cells). Bars, 100 µm or 25 µm (pSMAD3, right panels). Error bars indicate SE. *P
    Figure Legend Snippet: Microglial dysmaturation due to loss of TGFβ signaling is critically time dependent. (A) Diagram of Tgfbr2 gene inactivation and mutant analysis. Tamoxifen was injected at P1 for early induction in B or at P30 for adult induction in C. Mice were sacrificed and analyzed at indicated time points after tamoxifen injection. (B and C) Staining of cortical brain sections from Tgfbr2iΔMG and Cre + control littermates 120 d after tamoxifen administration following early (B) or adult (C) induction. Left panels: tdTomato recombination reporter (tdTom, red) marks all microglia and immature (APOE, green) and mature (P2RY12, blue) microglia markers to identify type A (dysmature) or type B microglia, respectively. Right panels (insets from Fig. S5): pSMAD3 (green) coimmunostaining reveals reduction in pSMAD3 staining intensity in type A (dysmature) microglia compared with type B microglia and controls. Percent (%) recombination (upper right graphs in B and C) based on % F4/80 + , CD45 + , CD11b + cells that are also tdTomato positive (cells isolated and analyzed by flow cytometry as in Fig. S3). Mean pSMAD3 per nucleus (lower right graphs in B and C) based on fluorescent intensity of individual recombined (tdTom + ) microglia coexpressing P2RY12 (blue, control and type B cells), or lacking P2RY12 expression (type A cells). Bars, 100 µm or 25 µm (pSMAD3, right panels). Error bars indicate SE. *P

    Techniques Used: Mutagenesis, Injection, Mouse Assay, Staining, Isolation, Flow Cytometry, Cytometry, Expressing

    59) Product Images from "A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture"

    Article Title: A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

    Journal: eLife

    doi: 10.7554/eLife.51276

    Changes in accessible chromatin, motif enrichment, and TF expression in primary brain ECs in culture. ( A ) Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) at or near Slco1c1 and Mfsd2a , BBB genes with reduced expression in cultured adult brain ECs (left panels), and Thbs1 and Cyr61 ( Ccn1 ), genes with enhanced expression in cultured adult brain ECs (right panels). Increases in transcript abundance are accompanied by increases in accessible chromatin near each gene (black arrows). Read counts are averaged over the independent replicates ( Figure 1—figure supplement 1 ). ( B ) PCA of ATAC-seq read density at all called ATAC-seq peaks in independent biological replicates from cultured adult brain ECs, acutely isolated adult and P7 brain ECs, and P7 liver, lung, and kidney ECs. Liver, lung, and kidney samples cluster at center right. The arrow points to cultured ECs. ( C ) Venn diagram summarizing the number of shared and distinct ATAC-seq peaks between acutely isolated and cultured adult brain ECs. ( D ) TF motif enrichment in ATAC-seq peaks that is specific to either acutely isolated adult brain ECs (left) or cultured adult brain ECs (right). Histograms of the -log 10 (p-value) for 414 TF DNA binding motifs that were tested for enrichment. The x-axis bin size is 1. The y-axis has been truncated. The vast majority of tested motifs have high p-values (i.e., low statistical significance) and thus are found in the first few bins. The statistically most significant TF motif families are labeled. Most TF families are represented by multiple closely related motifs, and only the motif with the lowest p-value for each family is labeled. The number of ATAC-seq peaks analyzed in ( D ) and ( E ) corresponds to the values shown in the Venn diagram in ( C ). ( E ) Enriched TF motifs identified by HOMER. The frequency of the indicated motifs is plotted as a function of distance from the center of the ATAC-seq peaks (from either acutely isolated or cultured adult brain ECs). Shown above each individual plot is the position weight matrix (PWM) of the enriched nucleotide sequence. The TF family that most closely matches the motif is indicated below the PWM. ( F ) Heatmap showing log 2 transformed TPMs for acutely isolated and cultured adult brain ECs for transcripts coding for a subset of TFs with the motifs shown in ( E ).
    Figure Legend Snippet: Changes in accessible chromatin, motif enrichment, and TF expression in primary brain ECs in culture. ( A ) Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) at or near Slco1c1 and Mfsd2a , BBB genes with reduced expression in cultured adult brain ECs (left panels), and Thbs1 and Cyr61 ( Ccn1 ), genes with enhanced expression in cultured adult brain ECs (right panels). Increases in transcript abundance are accompanied by increases in accessible chromatin near each gene (black arrows). Read counts are averaged over the independent replicates ( Figure 1—figure supplement 1 ). ( B ) PCA of ATAC-seq read density at all called ATAC-seq peaks in independent biological replicates from cultured adult brain ECs, acutely isolated adult and P7 brain ECs, and P7 liver, lung, and kidney ECs. Liver, lung, and kidney samples cluster at center right. The arrow points to cultured ECs. ( C ) Venn diagram summarizing the number of shared and distinct ATAC-seq peaks between acutely isolated and cultured adult brain ECs. ( D ) TF motif enrichment in ATAC-seq peaks that is specific to either acutely isolated adult brain ECs (left) or cultured adult brain ECs (right). Histograms of the -log 10 (p-value) for 414 TF DNA binding motifs that were tested for enrichment. The x-axis bin size is 1. The y-axis has been truncated. The vast majority of tested motifs have high p-values (i.e., low statistical significance) and thus are found in the first few bins. The statistically most significant TF motif families are labeled. Most TF families are represented by multiple closely related motifs, and only the motif with the lowest p-value for each family is labeled. The number of ATAC-seq peaks analyzed in ( D ) and ( E ) corresponds to the values shown in the Venn diagram in ( C ). ( E ) Enriched TF motifs identified by HOMER. The frequency of the indicated motifs is plotted as a function of distance from the center of the ATAC-seq peaks (from either acutely isolated or cultured adult brain ECs). Shown above each individual plot is the position weight matrix (PWM) of the enriched nucleotide sequence. The TF family that most closely matches the motif is indicated below the PWM. ( F ) Heatmap showing log 2 transformed TPMs for acutely isolated and cultured adult brain ECs for transcripts coding for a subset of TFs with the motifs shown in ( E ).

    Techniques Used: Expressing, RNA Sequencing Assay, Cell Culture, Isolation, Significance Assay, Binding Assay, Labeling, Sequencing, Transformation Assay

    WT and beta-catenin stabilized brain ECs in culture have nearly identical patterns of transcription and accessible chromatin. Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) for the same set of genes shown in Figure 2A for freshly isolated adult brain ECs, WT cultured adult brain ECs, and beta-catenin stabilized and cultured adult brain ECs. Read counts are averaged over the independent replicates. Arrows indicate ATAC-seq peaks that correlate with differential gene expression.
    Figure Legend Snippet: WT and beta-catenin stabilized brain ECs in culture have nearly identical patterns of transcription and accessible chromatin. Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) for the same set of genes shown in Figure 2A for freshly isolated adult brain ECs, WT cultured adult brain ECs, and beta-catenin stabilized and cultured adult brain ECs. Read counts are averaged over the independent replicates. Arrows indicate ATAC-seq peaks that correlate with differential gene expression.

    Techniques Used: RNA Sequencing Assay, Isolation, Cell Culture, Expressing

    In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.
    Figure Legend Snippet: In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.

    Techniques Used: In Vivo, FACS, Purification, Mouse Assay, RNA Sequencing Assay, Produced

    Characterization of primary brain EC cultures. ( A ) Primary brain ECs in culture, immunostained for CD31 (a pan-EC marker) and counterstained with DAPI. Merged image is shown in the right column. Scale bar: 100 um. ( B ) Genome browser images showing accessible chromatin (ATAC-seq; top) and transcript abundances (RNA-seq; bottom) for the Cd31 ( Pecam1 ) locus. Histograms show aligned read counts. Tracks in blue represent acutely isolated adult brain ECs and tracks in red represent cultured adult brain ECs. Each track represents an independent replicate (two replicates for acutely isolated adult brain ECs and six replicates for cultured adult brain ECs). All eight of the ATAC-seq histograms are at the same vertical scale and all eight of the RNA-seq histograms are at the same vertical scale. Bottom, intron-exon structure, with the arrow indicating the direction of transcription. ( C ) Heatmap showing pairwise Pearson correlations for RNA-seq TPM among acutely isolated adult brain ECs and cultured adult brain ECs for all protein-coding genes. Data are shown for the individual replicates, which were from four experiments, each with paired replicates: R1+R2 from brain ECs, and R1+R2, R3+R4, and R5+R6 from cultured ECs. ( D ) Scatter plots comparing cross-sample normalized RNA-seq read counts of all protein-coding genes between acutely isolated adult brain ECs and adult brain ECs cultured for 8 days. Each plot highlights the top 500 genes enriched in an identified cell type cluster from single-cell RNA-seq analysis of acutely isolated P7 brain ECs ( Sabbagh et al., 2018 ). The red arrow in the lower center plot points to a subset of mitotic genes that show enhanced expression in cultured ECs.
    Figure Legend Snippet: Characterization of primary brain EC cultures. ( A ) Primary brain ECs in culture, immunostained for CD31 (a pan-EC marker) and counterstained with DAPI. Merged image is shown in the right column. Scale bar: 100 um. ( B ) Genome browser images showing accessible chromatin (ATAC-seq; top) and transcript abundances (RNA-seq; bottom) for the Cd31 ( Pecam1 ) locus. Histograms show aligned read counts. Tracks in blue represent acutely isolated adult brain ECs and tracks in red represent cultured adult brain ECs. Each track represents an independent replicate (two replicates for acutely isolated adult brain ECs and six replicates for cultured adult brain ECs). All eight of the ATAC-seq histograms are at the same vertical scale and all eight of the RNA-seq histograms are at the same vertical scale. Bottom, intron-exon structure, with the arrow indicating the direction of transcription. ( C ) Heatmap showing pairwise Pearson correlations for RNA-seq TPM among acutely isolated adult brain ECs and cultured adult brain ECs for all protein-coding genes. Data are shown for the individual replicates, which were from four experiments, each with paired replicates: R1+R2 from brain ECs, and R1+R2, R3+R4, and R5+R6 from cultured ECs. ( D ) Scatter plots comparing cross-sample normalized RNA-seq read counts of all protein-coding genes between acutely isolated adult brain ECs and adult brain ECs cultured for 8 days. Each plot highlights the top 500 genes enriched in an identified cell type cluster from single-cell RNA-seq analysis of acutely isolated P7 brain ECs ( Sabbagh et al., 2018 ). The red arrow in the lower center plot points to a subset of mitotic genes that show enhanced expression in cultured ECs.

    Techniques Used: Marker, RNA Sequencing Assay, Isolation, Cell Culture, Expressing

    60) Product Images from "Targeting JNK pathway promotes human hematopoietic stem cell expansion"

    Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0072-8

    The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Flow Cytometry, Cell Counting

    JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p
    Figure Legend Snippet: JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p

    Techniques Used: Transplantation Assay, Injection, Two Tailed Test, Software

    JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p
    Figure Legend Snippet: JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p

    Techniques Used:

    JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1 . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p
    Figure Legend Snippet: JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1 . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p

    Techniques Used: In Vitro, Cell Culture, Concentration Assay

    JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p
    Figure Legend Snippet: JNK-IN-8-induced CD34 + cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Inhibition, Flow Cytometry, Cytometry, Transduction, shRNA, Transfection

    61) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    62) Product Images from "Deconstructing the principles of ductal network formation in the pancreas"

    Article Title: Deconstructing the principles of ductal network formation in the pancreas

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2002842

    Flux-based pruning of the VP14.5 networks. (A) The logarithm of the normalized flux at steady state of the E14.5 2 pancreas network. Thicker links indicate a higher flux. The highest flux is closest to the exit, with some interlinking nodes having very low flux. The links highlighted red are pruned by the pruning mechanism of least flux. (B) The pruning event's distance from the exit as pruning progresses for flux-based pruning and random pruning. (C) Average distance of all nodes to the exit scaled by the average distance between all nodes shown for the networks of E14.5, E18.5, the in silico pruned E14.5, and the E14.5 MST. Error bars represent SEM. Code files “Import_Experimental_data”, “DiffusionOnNetwork”, “PruneBasedOnFlux”, “SnapShot”, “ConvertToAdjMat”, “ConvertToAdjList”, “NetworkProp”, “NetworkShapes”, “FindTriangles”, “Remove_kinks” are provided in supporting information ( S1 Data ). E, embryonic day; MST, minimum spanning tree; ns, not significant; VP, ventral pancreas.
    Figure Legend Snippet: Flux-based pruning of the VP14.5 networks. (A) The logarithm of the normalized flux at steady state of the E14.5 2 pancreas network. Thicker links indicate a higher flux. The highest flux is closest to the exit, with some interlinking nodes having very low flux. The links highlighted red are pruned by the pruning mechanism of least flux. (B) The pruning event's distance from the exit as pruning progresses for flux-based pruning and random pruning. (C) Average distance of all nodes to the exit scaled by the average distance between all nodes shown for the networks of E14.5, E18.5, the in silico pruned E14.5, and the E14.5 MST. Error bars represent SEM. Code files “Import_Experimental_data”, “DiffusionOnNetwork”, “PruneBasedOnFlux”, “SnapShot”, “ConvertToAdjMat”, “ConvertToAdjList”, “NetworkProp”, “NetworkShapes”, “FindTriangles”, “Remove_kinks” are provided in supporting information ( S1 Data ). E, embryonic day; MST, minimum spanning tree; ns, not significant; VP, ventral pancreas.

    Techniques Used: In Silico, Microscale Thermophoresis

    63) Product Images from "CD177-mediated nanoparticle targeting of human and mouse neutrophils"

    Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200444

    Mouse C5aR1 siRNA and human C5aR1 siRNA pool result in receptor knockdown. CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.
    Figure Legend Snippet: Mouse C5aR1 siRNA and human C5aR1 siRNA pool result in receptor knockdown. CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.

    Techniques Used: Expressing, Transfection, Positive Control, Negative Control, Flow Cytometry, Cytometry

    Mouse C5aR1 ASO results in knockdown of mouse C5aR1-GFP in CHO transfectants. CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value
    Figure Legend Snippet: Mouse C5aR1 ASO results in knockdown of mouse C5aR1-GFP in CHO transfectants. CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value

    Techniques Used: Allele-specific Oligonucleotide, Expressing, Transfection, Flow Cytometry, Cytometry, Negative Control, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    64) Product Images from "A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture"

    Article Title: A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

    Journal: eLife

    doi: 10.7554/eLife.51276

    Changes in accessible chromatin, motif enrichment, and TF expression in primary brain ECs in culture. ( A ) Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) at or near Slco1c1 and Mfsd2a , BBB genes with reduced expression in cultured adult brain ECs (left panels), and Thbs1 and Cyr61 ( Ccn1 ), genes with enhanced expression in cultured adult brain ECs (right panels). Increases in transcript abundance are accompanied by increases in accessible chromatin near each gene (black arrows). Read counts are averaged over the independent replicates ( Figure 1—figure supplement 1 ). ( B ) PCA of ATAC-seq read density at all called ATAC-seq peaks in independent biological replicates from cultured adult brain ECs, acutely isolated adult and P7 brain ECs, and P7 liver, lung, and kidney ECs. Liver, lung, and kidney samples cluster at center right. The arrow points to cultured ECs. ( C ) Venn diagram summarizing the number of shared and distinct ATAC-seq peaks between acutely isolated and cultured adult brain ECs. ( D ) TF motif enrichment in ATAC-seq peaks that is specific to either acutely isolated adult brain ECs (left) or cultured adult brain ECs (right). Histograms of the -log 10 (p-value) for 414 TF DNA binding motifs that were tested for enrichment. The x-axis bin size is 1. The y-axis has been truncated. The vast majority of tested motifs have high p-values (i.e., low statistical significance) and thus are found in the first few bins. The statistically most significant TF motif families are labeled. Most TF families are represented by multiple closely related motifs, and only the motif with the lowest p-value for each family is labeled. The number of ATAC-seq peaks analyzed in ( D ) and ( E ) corresponds to the values shown in the Venn diagram in ( C ). ( E ) Enriched TF motifs identified by HOMER. The frequency of the indicated motifs is plotted as a function of distance from the center of the ATAC-seq peaks (from either acutely isolated or cultured adult brain ECs). Shown above each individual plot is the position weight matrix (PWM) of the enriched nucleotide sequence. The TF family that most closely matches the motif is indicated below the PWM. ( F ) Heatmap showing log 2 transformed TPMs for acutely isolated and cultured adult brain ECs for transcripts coding for a subset of TFs with the motifs shown in ( E ).
    Figure Legend Snippet: Changes in accessible chromatin, motif enrichment, and TF expression in primary brain ECs in culture. ( A ) Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) at or near Slco1c1 and Mfsd2a , BBB genes with reduced expression in cultured adult brain ECs (left panels), and Thbs1 and Cyr61 ( Ccn1 ), genes with enhanced expression in cultured adult brain ECs (right panels). Increases in transcript abundance are accompanied by increases in accessible chromatin near each gene (black arrows). Read counts are averaged over the independent replicates ( Figure 1—figure supplement 1 ). ( B ) PCA of ATAC-seq read density at all called ATAC-seq peaks in independent biological replicates from cultured adult brain ECs, acutely isolated adult and P7 brain ECs, and P7 liver, lung, and kidney ECs. Liver, lung, and kidney samples cluster at center right. The arrow points to cultured ECs. ( C ) Venn diagram summarizing the number of shared and distinct ATAC-seq peaks between acutely isolated and cultured adult brain ECs. ( D ) TF motif enrichment in ATAC-seq peaks that is specific to either acutely isolated adult brain ECs (left) or cultured adult brain ECs (right). Histograms of the -log 10 (p-value) for 414 TF DNA binding motifs that were tested for enrichment. The x-axis bin size is 1. The y-axis has been truncated. The vast majority of tested motifs have high p-values (i.e., low statistical significance) and thus are found in the first few bins. The statistically most significant TF motif families are labeled. Most TF families are represented by multiple closely related motifs, and only the motif with the lowest p-value for each family is labeled. The number of ATAC-seq peaks analyzed in ( D ) and ( E ) corresponds to the values shown in the Venn diagram in ( C ). ( E ) Enriched TF motifs identified by HOMER. The frequency of the indicated motifs is plotted as a function of distance from the center of the ATAC-seq peaks (from either acutely isolated or cultured adult brain ECs). Shown above each individual plot is the position weight matrix (PWM) of the enriched nucleotide sequence. The TF family that most closely matches the motif is indicated below the PWM. ( F ) Heatmap showing log 2 transformed TPMs for acutely isolated and cultured adult brain ECs for transcripts coding for a subset of TFs with the motifs shown in ( E ).

    Techniques Used: Expressing, RNA Sequencing Assay, Cell Culture, Isolation, Significance Assay, Binding Assay, Labeling, Sequencing, Transformation Assay

    WT and beta-catenin stabilized brain ECs in culture have nearly identical patterns of transcription and accessible chromatin. Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) for the same set of genes shown in Figure 2A for freshly isolated adult brain ECs, WT cultured adult brain ECs, and beta-catenin stabilized and cultured adult brain ECs. Read counts are averaged over the independent replicates. Arrows indicate ATAC-seq peaks that correlate with differential gene expression.
    Figure Legend Snippet: WT and beta-catenin stabilized brain ECs in culture have nearly identical patterns of transcription and accessible chromatin. Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) for the same set of genes shown in Figure 2A for freshly isolated adult brain ECs, WT cultured adult brain ECs, and beta-catenin stabilized and cultured adult brain ECs. Read counts are averaged over the independent replicates. Arrows indicate ATAC-seq peaks that correlate with differential gene expression.

    Techniques Used: RNA Sequencing Assay, Isolation, Cell Culture, Expressing

    Characterization of primary brain EC cultures. ( A ) Primary brain ECs in culture, immunostained for CD31 (a pan-EC marker) and counterstained with DAPI. Merged image is shown in the right column. Scale bar: 100 um. ( B ) Genome browser images showing accessible chromatin (ATAC-seq; top) and transcript abundances (RNA-seq; bottom) for the Cd31 ( Pecam1 ) locus. Histograms show aligned read counts. Tracks in blue represent acutely isolated adult brain ECs and tracks in red represent cultured adult brain ECs. Each track represents an independent replicate (two replicates for acutely isolated adult brain ECs and six replicates for cultured adult brain ECs). All eight of the ATAC-seq histograms are at the same vertical scale and all eight of the RNA-seq histograms are at the same vertical scale. Bottom, intron-exon structure, with the arrow indicating the direction of transcription. ( C ) Heatmap showing pairwise Pearson correlations for RNA-seq TPM among acutely isolated adult brain ECs and cultured adult brain ECs for all protein-coding genes. Data are shown for the individual replicates, which were from four experiments, each with paired replicates: R1+R2 from brain ECs, and R1+R2, R3+R4, and R5+R6 from cultured ECs. ( D ) Scatter plots comparing cross-sample normalized RNA-seq read counts of all protein-coding genes between acutely isolated adult brain ECs and adult brain ECs cultured for 8 days. Each plot highlights the top 500 genes enriched in an identified cell type cluster from single-cell RNA-seq analysis of acutely isolated P7 brain ECs ( Sabbagh et al., 2018 ). The red arrow in the lower center plot points to a subset of mitotic genes that show enhanced expression in cultured ECs.
    Figure Legend Snippet: Characterization of primary brain EC cultures. ( A ) Primary brain ECs in culture, immunostained for CD31 (a pan-EC marker) and counterstained with DAPI. Merged image is shown in the right column. Scale bar: 100 um. ( B ) Genome browser images showing accessible chromatin (ATAC-seq; top) and transcript abundances (RNA-seq; bottom) for the Cd31 ( Pecam1 ) locus. Histograms show aligned read counts. Tracks in blue represent acutely isolated adult brain ECs and tracks in red represent cultured adult brain ECs. Each track represents an independent replicate (two replicates for acutely isolated adult brain ECs and six replicates for cultured adult brain ECs). All eight of the ATAC-seq histograms are at the same vertical scale and all eight of the RNA-seq histograms are at the same vertical scale. Bottom, intron-exon structure, with the arrow indicating the direction of transcription. ( C ) Heatmap showing pairwise Pearson correlations for RNA-seq TPM among acutely isolated adult brain ECs and cultured adult brain ECs for all protein-coding genes. Data are shown for the individual replicates, which were from four experiments, each with paired replicates: R1+R2 from brain ECs, and R1+R2, R3+R4, and R5+R6 from cultured ECs. ( D ) Scatter plots comparing cross-sample normalized RNA-seq read counts of all protein-coding genes between acutely isolated adult brain ECs and adult brain ECs cultured for 8 days. Each plot highlights the top 500 genes enriched in an identified cell type cluster from single-cell RNA-seq analysis of acutely isolated P7 brain ECs ( Sabbagh et al., 2018 ). The red arrow in the lower center plot points to a subset of mitotic genes that show enhanced expression in cultured ECs.

    Techniques Used: Marker, RNA Sequencing Assay, Isolation, Cell Culture, Expressing

    65) Product Images from "Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration"

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2016.12.015

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Figure Legend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .
    Figure Legend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Techniques Used: RNA Sequencing Assay, Functional Assay

    66) Product Images from "Impaired αVβ8 and TGFβ signaling lead to microglial dysmaturation and neuromotor dysfunction"

    Article Title: Impaired αVβ8 and TGFβ signaling lead to microglial dysmaturation and neuromotor dysfunction

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20181290

    Microglial dysmaturation due to loss of TGFβ signaling is critically time dependent. (A) Diagram of Tgfbr2 gene inactivation and mutant analysis. Tamoxifen was injected at P1 for early induction in B or at P30 for adult induction in C. Mice were sacrificed and analyzed at indicated time points after tamoxifen injection. (B and C) Staining of cortical brain sections from Tgfbr2iΔMG and Cre + control littermates 120 d after tamoxifen administration following early (B) or adult (C) induction. Left panels: tdTomato recombination reporter (tdTom, red) marks all microglia and immature (APOE, green) and mature (P2RY12, blue) microglia markers to identify type A (dysmature) or type B microglia, respectively. Right panels (insets from Fig. S5): pSMAD3 (green) coimmunostaining reveals reduction in pSMAD3 staining intensity in type A (dysmature) microglia compared with type B microglia and controls. Percent (%) recombination (upper right graphs in B and C) based on % F4/80 + , CD45 + , CD11b + cells that are also tdTomato positive (cells isolated and analyzed by flow cytometry as in Fig. S3). Mean pSMAD3 per nucleus (lower right graphs in B and C) based on fluorescent intensity of individual recombined (tdTom + ) microglia coexpressing P2RY12 (blue, control and type B cells), or lacking P2RY12 expression (type A cells). Bars, 100 µm or 25 µm (pSMAD3, right panels). Error bars indicate SE. *P
    Figure Legend Snippet: Microglial dysmaturation due to loss of TGFβ signaling is critically time dependent. (A) Diagram of Tgfbr2 gene inactivation and mutant analysis. Tamoxifen was injected at P1 for early induction in B or at P30 for adult induction in C. Mice were sacrificed and analyzed at indicated time points after tamoxifen injection. (B and C) Staining of cortical brain sections from Tgfbr2iΔMG and Cre + control littermates 120 d after tamoxifen administration following early (B) or adult (C) induction. Left panels: tdTomato recombination reporter (tdTom, red) marks all microglia and immature (APOE, green) and mature (P2RY12, blue) microglia markers to identify type A (dysmature) or type B microglia, respectively. Right panels (insets from Fig. S5): pSMAD3 (green) coimmunostaining reveals reduction in pSMAD3 staining intensity in type A (dysmature) microglia compared with type B microglia and controls. Percent (%) recombination (upper right graphs in B and C) based on % F4/80 + , CD45 + , CD11b + cells that are also tdTomato positive (cells isolated and analyzed by flow cytometry as in Fig. S3). Mean pSMAD3 per nucleus (lower right graphs in B and C) based on fluorescent intensity of individual recombined (tdTom + ) microglia coexpressing P2RY12 (blue, control and type B cells), or lacking P2RY12 expression (type A cells). Bars, 100 µm or 25 µm (pSMAD3, right panels). Error bars indicate SE. *P

    Techniques Used: Mutagenesis, Injection, Mouse Assay, Staining, Isolation, Flow Cytometry, Cytometry, Expressing

    67) Product Images from "Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1"

    Article Title: Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01738-3

    Vascular alterations after intraocular VEGF-A injection. a Morphology of IB4-stained P6 wild-type retinal vessels at 4 h after administration of human VEGF-A165 (0.5 µl at a concentration of 5 μg μl −1 ). Note blunt appearance of the vessel front after VEGF-A injection but not for vehicle (PBS) control. Scale bar, 200 µm. b Quantitation of sprouts and filopodia at the front of the P6 vessel plexus after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test. c PDGFRβ+ (green) pericytes are unaffected by short-term VEGF-A administration, whereas VEGFR2 immunosignals (white) are increased in IB4+ (red) ECs (arrowheads). Images shown correspond to insets in a . Scale bar, 100 µm. d Quantitation of VEGFR2 immunosignals intensity in the peripheral plexus of P6 retinas after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test. e Confocal images showing increased Esm1 immunostaining (white) in IB4+ (red) ECs in the peripheral plexus (arrowheads) after VEGF-A injection in P6 pups. Scale bar, 200 µm. f VEGF-A165 injection-mediated increase of Esm1 immunosignals (normalized to IB4+ EC area) in the peripheral capillary plexus but not at the edge of the angiogenic front in comparison to PBS-injected controls at P6. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. g Short-term VEGF-A165 administration leads to clustering of Erg1+ (green) and IB4+ (red) ECs, as indicated, in thick sprout-like structures of P6 retinas. Panels in the center and on the right (scale bar, 20 µm) show higher magnification of the insets on the left (scale bar, 100 µm). Dashed lines in panels on the right outline IB4+ vessels. h Quantitation of EC density in the leading front vessel and emerging sprouts of the P6 angiogenic front after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test
    Figure Legend Snippet: Vascular alterations after intraocular VEGF-A injection. a Morphology of IB4-stained P6 wild-type retinal vessels at 4 h after administration of human VEGF-A165 (0.5 µl at a concentration of 5 μg μl −1 ). Note blunt appearance of the vessel front after VEGF-A injection but not for vehicle (PBS) control. Scale bar, 200 µm. b Quantitation of sprouts and filopodia at the front of the P6 vessel plexus after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test. c PDGFRβ+ (green) pericytes are unaffected by short-term VEGF-A administration, whereas VEGFR2 immunosignals (white) are increased in IB4+ (red) ECs (arrowheads). Images shown correspond to insets in a . Scale bar, 100 µm. d Quantitation of VEGFR2 immunosignals intensity in the peripheral plexus of P6 retinas after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test. e Confocal images showing increased Esm1 immunostaining (white) in IB4+ (red) ECs in the peripheral plexus (arrowheads) after VEGF-A injection in P6 pups. Scale bar, 200 µm. f VEGF-A165 injection-mediated increase of Esm1 immunosignals (normalized to IB4+ EC area) in the peripheral capillary plexus but not at the edge of the angiogenic front in comparison to PBS-injected controls at P6. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. g Short-term VEGF-A165 administration leads to clustering of Erg1+ (green) and IB4+ (red) ECs, as indicated, in thick sprout-like structures of P6 retinas. Panels in the center and on the right (scale bar, 20 µm) show higher magnification of the insets on the left (scale bar, 100 µm). Dashed lines in panels on the right outline IB4+ vessels. h Quantitation of EC density in the leading front vessel and emerging sprouts of the P6 angiogenic front after injection of VEGF-A165 or vehicle control. Error bars, s.e.m. p -values, Student’s t -test

    Techniques Used: Injection, Staining, Concentration Assay, Quantitation Assay, Immunostaining

    Expression of sFlt1 in pericytes at the angiogenic front. a Maximum intensity projections of confocal images from P6 retinas of the Hey1 -GFP transgenic reporter mouse model stained for GFP (green), PDGFRβ+ (white) and IB4 (red). Images of the first row show enrichment of Hey1 -GFP+, PDGFRβ+ perivascular cells in the angiogenic front in comparison to mural cells covering the remodeling central plexus around veins (middle row) and arteries (bottom row). Note strong expression of Hey1 -GFP reporter in arterial ECs (bottom row). Scale bar , 50 µm. b , Quantitation of Pdgfrb expression by qPCR in P6 PDGFRβ+ retinal pericytes sorted based on GFP expression in comparison to whole-retina single-cell suspension (input). Note significant enrichment of Pdgfrb in both (GFP+ and GFP−) pericyte fractions and higher expression in the Hey1 -GFP+ subset. Error bars, s.e.m. p -values, Kruskal–Wallis and Dunn’s multiple comparison test. NS, not statistically significant. c Quantitation of sFlt1 expression by qPCR in sorted P6 retinal pericytes in comparison to whole-retina single-cell suspension (input). Note significant enrichment of sFlt1 expression in Hey1 -GFP+ pericytes in comparison to input and GFP- pericytes. Error bars, s.e.m. p -values, one-way ANOVA and Tukey’s multiple comparison test. NS, not statistically significant
    Figure Legend Snippet: Expression of sFlt1 in pericytes at the angiogenic front. a Maximum intensity projections of confocal images from P6 retinas of the Hey1 -GFP transgenic reporter mouse model stained for GFP (green), PDGFRβ+ (white) and IB4 (red). Images of the first row show enrichment of Hey1 -GFP+, PDGFRβ+ perivascular cells in the angiogenic front in comparison to mural cells covering the remodeling central plexus around veins (middle row) and arteries (bottom row). Note strong expression of Hey1 -GFP reporter in arterial ECs (bottom row). Scale bar , 50 µm. b , Quantitation of Pdgfrb expression by qPCR in P6 PDGFRβ+ retinal pericytes sorted based on GFP expression in comparison to whole-retina single-cell suspension (input). Note significant enrichment of Pdgfrb in both (GFP+ and GFP−) pericyte fractions and higher expression in the Hey1 -GFP+ subset. Error bars, s.e.m. p -values, Kruskal–Wallis and Dunn’s multiple comparison test. NS, not statistically significant. c Quantitation of sFlt1 expression by qPCR in sorted P6 retinal pericytes in comparison to whole-retina single-cell suspension (input). Note significant enrichment of sFlt1 expression in Hey1 -GFP+ pericytes in comparison to input and GFP- pericytes. Error bars, s.e.m. p -values, one-way ANOVA and Tukey’s multiple comparison test. NS, not statistically significant

    Techniques Used: Expressing, Transgenic Assay, Staining, Quantitation Assay, Real-time Polymerase Chain Reaction

    Inactivation of Flt1 in PDGFRβ+ cells. a Experimental scheme of tamoxifen administration for the generation of Flt1 iPC mutants. b P6 control, Flt1 iPC/+ and Flt1 iPC retinas stained with isolectin B4 (IB4). Dashed circles indicate vessel-covered (yellow) and peripheral avascular (white) areas in the overview pictures (top). Scale bar, 500 µm. c Quantitation of body weight and radial outgrowth of the retinal vasculature in control, Flt1 iPC/+ and Flt1 iPC P6 pups. Error bars, s.e.m. p -values, one-way ANOVA. NS, not statistically significant. d Confocal images of the IB4-stained P6 control, Flt1 iPC/+ and Flt1 iPC retinal angiogenic front illustrating differences in sprout number and morphology. Scale bar, 100 µm. e Quantitation of sprouts and filopodia in P6 control, Flt1 iPC/+ and Flt1 iPC retinas. Error bars, s.e.m. p -values, one-way ANOVA and Tukey’s multiple comparison test. NS, not statistically significant. f Confocal images of IB4 (red), Erg1 (green) and VEGFR2 (white) stained P6 retinas highlighting the accumulation of EC nuclei and enhanced VEGFR2 immunosignals (arrowheads) in Flt1 iPC sprouts. Vessels are outlined by dashed lines on the right panel. Scale bar, 100 µm. g Quantitation of EC proliferation (EdU+ Erg1+) at the angiogenic front, EC density in sprouts and leading front vessel and VEGFR2 immunosignals intensity in the angiogenic front of control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. h Esm1 (white) expression (arrowheads) in the angiogenic front (IB4+, red, first two columns) and detection of desmin+ pericytes (green, third column) in P6 control and Flt1 iPC retinas. Scale bar, 100 µm. i Quantitation of Esm1+ proportion relative to vascular area (IB4+) in the angiogenic front of control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. j Confocal images of P6 retinas stained for NG2 (green) and IB4 (red) showing no significant changes in pericyte coverage in the front (first two columns) or the remodeling plexus around veins (v) or arteries (a) (last two columns). Scale bar, 100 µm. k , l Quantitation of pericyte coverage k and relative gene expression by qPCR on whole lysates l in control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant
    Figure Legend Snippet: Inactivation of Flt1 in PDGFRβ+ cells. a Experimental scheme of tamoxifen administration for the generation of Flt1 iPC mutants. b P6 control, Flt1 iPC/+ and Flt1 iPC retinas stained with isolectin B4 (IB4). Dashed circles indicate vessel-covered (yellow) and peripheral avascular (white) areas in the overview pictures (top). Scale bar, 500 µm. c Quantitation of body weight and radial outgrowth of the retinal vasculature in control, Flt1 iPC/+ and Flt1 iPC P6 pups. Error bars, s.e.m. p -values, one-way ANOVA. NS, not statistically significant. d Confocal images of the IB4-stained P6 control, Flt1 iPC/+ and Flt1 iPC retinal angiogenic front illustrating differences in sprout number and morphology. Scale bar, 100 µm. e Quantitation of sprouts and filopodia in P6 control, Flt1 iPC/+ and Flt1 iPC retinas. Error bars, s.e.m. p -values, one-way ANOVA and Tukey’s multiple comparison test. NS, not statistically significant. f Confocal images of IB4 (red), Erg1 (green) and VEGFR2 (white) stained P6 retinas highlighting the accumulation of EC nuclei and enhanced VEGFR2 immunosignals (arrowheads) in Flt1 iPC sprouts. Vessels are outlined by dashed lines on the right panel. Scale bar, 100 µm. g Quantitation of EC proliferation (EdU+ Erg1+) at the angiogenic front, EC density in sprouts and leading front vessel and VEGFR2 immunosignals intensity in the angiogenic front of control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. h Esm1 (white) expression (arrowheads) in the angiogenic front (IB4+, red, first two columns) and detection of desmin+ pericytes (green, third column) in P6 control and Flt1 iPC retinas. Scale bar, 100 µm. i Quantitation of Esm1+ proportion relative to vascular area (IB4+) in the angiogenic front of control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. j Confocal images of P6 retinas stained for NG2 (green) and IB4 (red) showing no significant changes in pericyte coverage in the front (first two columns) or the remodeling plexus around veins (v) or arteries (a) (last two columns). Scale bar, 100 µm. k , l Quantitation of pericyte coverage k and relative gene expression by qPCR on whole lysates l in control and Flt1 iPC P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant

    Techniques Used: Staining, Quantitation Assay, Expressing, Real-time Polymerase Chain Reaction

    Temporally controlled pericyte depletion. a Experimental scheme of tamoxifen and diphtheria toxin (DT) administration to postnatal DTR iPC mice. b Confocal images showing P6 retina whole-mounts stained with isolectin B4 (IB4, red), collagen IV (colIV, white) and PDGFRβ (green). Note reduction of PDGFRβ+ cells at the DTR iPC angiogenic front. Scale bar, 100 µm. c High-magnification confocal images of the desmin (green) and IB4 (red) stained peripheral vasculature in the P6 DTR iPC and littermate control retina. Note extended morphology of desmin+ intermediate filaments in residual DTR iPC perivascular cells. Scale bar, 100 µm. d Maximum intensity projections of P6 control and DTR iPC retinas stained for NG2 (green) and IB4 (red). Images in the two right columns show higher magnifications of insets representing angiogenic front (AF, white dashed squares) and central plexus (CP, yellow-dashed squares), respectively. Scale bar, 200 µm (images on the left) and 100 µm (higher magnifications). e Quantitation of pericyte (PC) coverage in the control and DTR iPC angiogenic front and capillary plexus, as indicated. Error bars, s.e.m. p -values, Student’s t -test
    Figure Legend Snippet: Temporally controlled pericyte depletion. a Experimental scheme of tamoxifen and diphtheria toxin (DT) administration to postnatal DTR iPC mice. b Confocal images showing P6 retina whole-mounts stained with isolectin B4 (IB4, red), collagen IV (colIV, white) and PDGFRβ (green). Note reduction of PDGFRβ+ cells at the DTR iPC angiogenic front. Scale bar, 100 µm. c High-magnification confocal images of the desmin (green) and IB4 (red) stained peripheral vasculature in the P6 DTR iPC and littermate control retina. Note extended morphology of desmin+ intermediate filaments in residual DTR iPC perivascular cells. Scale bar, 100 µm. d Maximum intensity projections of P6 control and DTR iPC retinas stained for NG2 (green) and IB4 (red). Images in the two right columns show higher magnifications of insets representing angiogenic front (AF, white dashed squares) and central plexus (CP, yellow-dashed squares), respectively. Scale bar, 200 µm (images on the left) and 100 µm (higher magnifications). e Quantitation of pericyte (PC) coverage in the control and DTR iPC angiogenic front and capillary plexus, as indicated. Error bars, s.e.m. p -values, Student’s t -test

    Techniques Used: Mouse Assay, Staining, Quantitation Assay

    Altered endothelial sprouting after pericyte depletion. a Isolectin B4-stained P6 control and DTR iPC retina whole-mounts. Dashed circles indicate vessel-covered (yellow) and peripheral avascular (white) areas, respectively. Scale bar, 500 µm. b , c Quantitation of P6 control and DTR iPC body weight b and radial outgrowth of the retinal vasculature c . Error bars, s.e.m. p -values, Student’s t -test. d Confocal images of IB4 (white/red) and collagen IV (colIV, blue) showing the reduction of branch points but no increase in IB4- colIV+ empty matrix sleeves in DTR iPC P6 retinas. Scale bar, 200 µm (top panels) and 50 µm (bottom). e , f Quantitation of branch points e and empty matrix sleeves f in P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. g Maximum intensity projection of the IB4-stained P6 control and DTR iPC retinal angiogenic front illustrating differences in sprout number, morphology and filopodia number after pericyte depletion. Scale bar, 50 µm. h , i Quantitation of sprouts h and filopodia number i in the P6 control and DTR iPC angiogenic front. Error bars, s.e.m. p -values, Student’s t -test
    Figure Legend Snippet: Altered endothelial sprouting after pericyte depletion. a Isolectin B4-stained P6 control and DTR iPC retina whole-mounts. Dashed circles indicate vessel-covered (yellow) and peripheral avascular (white) areas, respectively. Scale bar, 500 µm. b , c Quantitation of P6 control and DTR iPC body weight b and radial outgrowth of the retinal vasculature c . Error bars, s.e.m. p -values, Student’s t -test. d Confocal images of IB4 (white/red) and collagen IV (colIV, blue) showing the reduction of branch points but no increase in IB4- colIV+ empty matrix sleeves in DTR iPC P6 retinas. Scale bar, 200 µm (top panels) and 50 µm (bottom). e , f Quantitation of branch points e and empty matrix sleeves f in P6 retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. g Maximum intensity projection of the IB4-stained P6 control and DTR iPC retinal angiogenic front illustrating differences in sprout number, morphology and filopodia number after pericyte depletion. Scale bar, 50 µm. h , i Quantitation of sprouts h and filopodia number i in the P6 control and DTR iPC angiogenic front. Error bars, s.e.m. p -values, Student’s t -test

    Techniques Used: Staining, Quantitation Assay

    Endothelial changes after pericyte depletion. a – f Maximum intensity projection of confocal images from control and DTR iPC P6 retinas stained for IB4 (red) in combination with VEGF-A a , VEGFR2 b , VEGFR3 c , Tie2 d , Esm1 e , and Dll4 f (all in white), as indicated. Note local increase of VEGFR2, VEGFR3, and Esm1 (arrowheads in b , c , e ) but not Tie2 or VEGF-A at the edge of the vessel plexus. Dll4 expression in DTR iPC sprouts is increased in some regions (arrowheads) but absent in others (arrows). Scale bar, 100 µm. g – j Quantitation of VEGF-A immunosignals area and intensity g , signal intensity for VEGFR2 h and VEGFR3 i and proportion of Esm1+ area with respect to vascular area j in the P6 control and DTR iPC angiogenic front. Error bars, s.e.m. p -values, Student’s t -test
    Figure Legend Snippet: Endothelial changes after pericyte depletion. a – f Maximum intensity projection of confocal images from control and DTR iPC P6 retinas stained for IB4 (red) in combination with VEGF-A a , VEGFR2 b , VEGFR3 c , Tie2 d , Esm1 e , and Dll4 f (all in white), as indicated. Note local increase of VEGFR2, VEGFR3, and Esm1 (arrowheads in b , c , e ) but not Tie2 or VEGF-A at the edge of the vessel plexus. Dll4 expression in DTR iPC sprouts is increased in some regions (arrowheads) but absent in others (arrows). Scale bar, 100 µm. g – j Quantitation of VEGF-A immunosignals area and intensity g , signal intensity for VEGFR2 h and VEGFR3 i and proportion of Esm1+ area with respect to vascular area j in the P6 control and DTR iPC angiogenic front. Error bars, s.e.m. p -values, Student’s t -test

    Techniques Used: Staining, Expressing, Quantitation Assay

    Pericytes control endothelial behavior in angiogenesis. a Confocal images showing proliferating cells (EdU, blue), IB4 (red) and EC nuclei (Erg1, green) in the P6 control and DTR iPC retinal angiogenic front, as indicated. Images on the right show higher magnification of boxed insets in left column. Dashed lines outline the angiogenic leading vessel from where the sprouts emerge (second column) or vessel shape (fourth column) at the edge of the vascular plexus. Scale bar, 100 µm (left panels) and 50 µm (higher magnifications). b – d Quantitation of total b and regional EC proliferation c as well as EC density d within the peripheral vascular plexus of P6 control and DTR iPC retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. e High magnification of IB4 (red) and Erg1 (green) stained sprouts highlighting the accumulation of EC nuclei in P6 DTR iPC but not control sprouts. Scale bar, 50 µm. f Confocal images showing IB4 (red), NG2+ pericytes (green) and vessel lumen (ICAM2, white) in the control and DTR iPC angiogenic front of P6 retinas. Images on the right show higher magnification of boxed insets in the second column. Note that IB4 signal covers a larger area than the apical ICAM2 immunostaining (arrowheads in third column). Scale bar, 100 µm (left panels) and 50 µm (higher magnifications)
    Figure Legend Snippet: Pericytes control endothelial behavior in angiogenesis. a Confocal images showing proliferating cells (EdU, blue), IB4 (red) and EC nuclei (Erg1, green) in the P6 control and DTR iPC retinal angiogenic front, as indicated. Images on the right show higher magnification of boxed insets in left column. Dashed lines outline the angiogenic leading vessel from where the sprouts emerge (second column) or vessel shape (fourth column) at the edge of the vascular plexus. Scale bar, 100 µm (left panels) and 50 µm (higher magnifications). b – d Quantitation of total b and regional EC proliferation c as well as EC density d within the peripheral vascular plexus of P6 control and DTR iPC retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. e High magnification of IB4 (red) and Erg1 (green) stained sprouts highlighting the accumulation of EC nuclei in P6 DTR iPC but not control sprouts. Scale bar, 50 µm. f Confocal images showing IB4 (red), NG2+ pericytes (green) and vessel lumen (ICAM2, white) in the control and DTR iPC angiogenic front of P6 retinas. Images on the right show higher magnification of boxed insets in the second column. Note that IB4 signal covers a larger area than the apical ICAM2 immunostaining (arrowheads in third column). Scale bar, 100 µm (left panels) and 50 µm (higher magnifications)

    Techniques Used: Quantitation Assay, Staining, Immunostaining

    68) Product Images from "Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates"

    Article Title: Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41505-6

    Differential expression of transcripts from RNA-SEQ. ( a – d ) Volcano plots comparing fold-change and p-value of each identified transcript accession for ( a , b ) U87 mg and ( c , d ) DAOY cellular aggregates for samples exposed to 250 V/m dcEFs for ( a , c ) 2 h, or ( b , d ) 8 h compared 0 h, unexposed controls. Significance threshold is set to FDR
    Figure Legend Snippet: Differential expression of transcripts from RNA-SEQ. ( a – d ) Volcano plots comparing fold-change and p-value of each identified transcript accession for ( a , b ) U87 mg and ( c , d ) DAOY cellular aggregates for samples exposed to 250 V/m dcEFs for ( a , c ) 2 h, or ( b , d ) 8 h compared 0 h, unexposed controls. Significance threshold is set to FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    69) Product Images from "Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions"

    Article Title: Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0900-16.2016

    Intracellular localization of U18-induced cholesterol accumulation. A , Phase-contrast micrograph showing somata and neurites of RGCs purified by immunopanning from 1-week-old rats and cultured for 4 d under serum- and glia-free conditions. B , Fluorescence micrographs of RGCs treated without [control (Con)] or with U18 (2.5 μg/ml for 48 h) and stained with filipin. C , Numbers of filipin-positive puncta in somata of RGCs (top; for 0, 0.1, 0.5, 1.0, and 2.5 μg/ml, n = 638, 557, 594, 451, and 678 cells, respectively; 2–3 preparations) and percentage of surviving RGCs (bottom) treated with U18 at indicated concentrations for 48 h. Viability values were normalized to untreated control cultures (3–4 preparations). Inset, False-color fluorescence micrograph showing living (green) and dead (red) RGCs from an untreated control culture. D , Numbers (left axis, white boxes; Con, n = 311 cells; GCM, n = 328 cells) and intensities of filipin-positive puncta (right axis, blue boxes; Con, n = 284 cells; GCM, n = 325 cells; values divided by 1000) in somata of RGCs treated with U18 in the presence or absence of GCM (3 preparations; Mann–Whitney U test). ** p
    Figure Legend Snippet: Intracellular localization of U18-induced cholesterol accumulation. A , Phase-contrast micrograph showing somata and neurites of RGCs purified by immunopanning from 1-week-old rats and cultured for 4 d under serum- and glia-free conditions. B , Fluorescence micrographs of RGCs treated without [control (Con)] or with U18 (2.5 μg/ml for 48 h) and stained with filipin. C , Numbers of filipin-positive puncta in somata of RGCs (top; for 0, 0.1, 0.5, 1.0, and 2.5 μg/ml, n = 638, 557, 594, 451, and 678 cells, respectively; 2–3 preparations) and percentage of surviving RGCs (bottom) treated with U18 at indicated concentrations for 48 h. Viability values were normalized to untreated control cultures (3–4 preparations). Inset, False-color fluorescence micrograph showing living (green) and dead (red) RGCs from an untreated control culture. D , Numbers (left axis, white boxes; Con, n = 311 cells; GCM, n = 328 cells) and intensities of filipin-positive puncta (right axis, blue boxes; Con, n = 284 cells; GCM, n = 325 cells; values divided by 1000) in somata of RGCs treated with U18 in the presence or absence of GCM (3 preparations; Mann–Whitney U test). ** p

    Techniques Used: Purification, Cell Culture, Fluorescence, Staining, MANN-WHITNEY

    Gene expression profiling by RNAseq of RGCs acutely purified from wild-type and NPC1-deficient mice. A , Left, false-color micrographs of RGCs acutely purified from 1-week-old mice by immunopanning and subjected to nuclear staining with DAPI (blue) and to immunocytochemical staining for Thy1 (green). Scale bar, 20 μm. Right, percentage of Thy1+ cells after isolation from retinae of 1-week-old ( n = 9) and 2-week-old mice ( n = 5; t test). Immunopanning delivered ∼73,000 ± 8,000 Thy1-positive cells per 1-week-old mouse ( n = 5). B , Quantity of total RNA in purified RGCs per mouse ( n = 4 mice per genotype corresponding to biological replicates; t test). C , Heat map of Pearson's correlation coefficient showing the reproducibility of transcript counts among biological replicates. D , First factorial plane resulting from a correspondence analysis of variance-stabilized data with the x -axis and y -axis explaining 23 and 22% of the variability of the whole dataset, respectively. E , Mean fold changes of transcript counts in RGCs from 1-week-old mutant mice compared to wild-type littermates plotted against normalized mean counts of each transcript as revealed by RNAseq. Red dots indicate genes with an adjusted p value of
    Figure Legend Snippet: Gene expression profiling by RNAseq of RGCs acutely purified from wild-type and NPC1-deficient mice. A , Left, false-color micrographs of RGCs acutely purified from 1-week-old mice by immunopanning and subjected to nuclear staining with DAPI (blue) and to immunocytochemical staining for Thy1 (green). Scale bar, 20 μm. Right, percentage of Thy1+ cells after isolation from retinae of 1-week-old ( n = 9) and 2-week-old mice ( n = 5; t test). Immunopanning delivered ∼73,000 ± 8,000 Thy1-positive cells per 1-week-old mouse ( n = 5). B , Quantity of total RNA in purified RGCs per mouse ( n = 4 mice per genotype corresponding to biological replicates; t test). C , Heat map of Pearson's correlation coefficient showing the reproducibility of transcript counts among biological replicates. D , First factorial plane resulting from a correspondence analysis of variance-stabilized data with the x -axis and y -axis explaining 23 and 22% of the variability of the whole dataset, respectively. E , Mean fold changes of transcript counts in RGCs from 1-week-old mutant mice compared to wild-type littermates plotted against normalized mean counts of each transcript as revealed by RNAseq. Red dots indicate genes with an adjusted p value of

    Techniques Used: Expressing, Purification, Mouse Assay, Staining, Isolation, Mutagenesis

    70) Product Images from "Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development"

    Article Title: Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07451-z

    Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR
    Figure Legend Snippet: Mouse Smarcd3 -F6 enhancer labels early cardiac progenitors in zebrafish. a Generation of Tg(Smarcd3-F6:EGFP) zebrafish line b In-situ hybridization against gfp transcripts on Tg(Smarcd3-F6:EGFP) transgenic embryos. Smarcd3-F6 enhancer marks lateral margins (arrowheads) during gastrulation and ALPM regions (arrows) after gastrulation. c Native GFP expression in Tg(Smarcd3-F6:EGFP) embryos at 10 hpf. Embryos are shown in lateral views. d Immunostaining of GFP and ZsYellow on Tg(Smarcd3-F6:EGFP) and Tg(nkx2.5:ZsYellow) double transgenic embryos. Cells expressing ZsYellow were marked by GFP as well. e Workflow of mRNA-seq and ATAC-seq experiments. f Volcano plot showing genes differentially expressed between Smarcd3-F6 :GFP+ and Smarcd3-F6 :GFP- populations (FDR

    Techniques Used: In Situ Hybridization, Transgenic Assay, Expressing, Immunostaining

    71) Product Images from "Adipocyte cannabinoid receptor CB1 regulates energy homeostasis and alternatively activated macrophages"

    Article Title: Adipocyte cannabinoid receptor CB1 regulates energy homeostasis and alternatively activated macrophages

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI83626

    CB1 deletion in adipocytes affects caloric intake and EE and promotes alternative macrophage activation. ( A ) Daily caloric intake (in kJ) of Ati-CB1–WT (SD fed, n = 11; HFD fed, n = 21) and Ati-CB1–KO mice (SD fed, n = 17; HFD fed, n = 27) on SD and HFD. ( B ) Pair-feeding experiment. Body weight curves of Ati-CB1–WT ( n = 8) and Ati-CB1–KO ( n = 13) on HFD. Tissue NE turnover in EF ( C ), SF ( D ), and BAT ( E ) from Ati-CB1–WT ( n = 3) and Ati-CB1–KO mice ( n = 4) on HFD. ( F ) EE in Ati-CB1–WT ( n = 16) and Ati-CB1–KO mice ( n = 13) on HFD. ( G ) Ambulatory activity during indirect calorimetry recording in Ati-CB1–WT ( n = 16) and Ati-CB1–KO ( n = 13) on HFD. ( H – J ) Gene expression analysis (relative units) of markers for alternatively activated macrophages ( Mrc1 , Clec10a ) in EF, SF, and BAT from Ati-CB1–WT and Ati-CB1–KO mice on HFD ( n = 11–12). ( K ) Protein markers for alternatively activated macrophages (CD206 and CD301) were monitored by flow cytometry in EF from Ati-CB1–WT and Ati-CB1–KO mice on HFD ( n = 11–12). ( L ) Gene expression (relative units) of the catecholamine-synthesizing enzymes ( Th , Dbh , Ddc ) and alternatively activated macrophage markers ( Mrc1 , Clec10a ) in CD11b + F4/80 + -sorted ATMs from WAT of Ati-CB1–WT ( n = 6–11) and Ati-CB1–KO ( n = 5–11) mice on HFD. Dbh mRNA levels were measured by ddPCR analysis. ( M ) TH protein levels were measured by flow cytometry in CD11b + F4/80 + -sorted ATMs (left) and CD301 + cells (M2 macrophages, right) in WAT from Ati-CB1–WT ( n = 9) and Ati-CB1–KO ( n = 9) mice on HFD. Data are shown as mean ± SEM. * P
    Figure Legend Snippet: CB1 deletion in adipocytes affects caloric intake and EE and promotes alternative macrophage activation. ( A ) Daily caloric intake (in kJ) of Ati-CB1–WT (SD fed, n = 11; HFD fed, n = 21) and Ati-CB1–KO mice (SD fed, n = 17; HFD fed, n = 27) on SD and HFD. ( B ) Pair-feeding experiment. Body weight curves of Ati-CB1–WT ( n = 8) and Ati-CB1–KO ( n = 13) on HFD. Tissue NE turnover in EF ( C ), SF ( D ), and BAT ( E ) from Ati-CB1–WT ( n = 3) and Ati-CB1–KO mice ( n = 4) on HFD. ( F ) EE in Ati-CB1–WT ( n = 16) and Ati-CB1–KO mice ( n = 13) on HFD. ( G ) Ambulatory activity during indirect calorimetry recording in Ati-CB1–WT ( n = 16) and Ati-CB1–KO ( n = 13) on HFD. ( H – J ) Gene expression analysis (relative units) of markers for alternatively activated macrophages ( Mrc1 , Clec10a ) in EF, SF, and BAT from Ati-CB1–WT and Ati-CB1–KO mice on HFD ( n = 11–12). ( K ) Protein markers for alternatively activated macrophages (CD206 and CD301) were monitored by flow cytometry in EF from Ati-CB1–WT and Ati-CB1–KO mice on HFD ( n = 11–12). ( L ) Gene expression (relative units) of the catecholamine-synthesizing enzymes ( Th , Dbh , Ddc ) and alternatively activated macrophage markers ( Mrc1 , Clec10a ) in CD11b + F4/80 + -sorted ATMs from WAT of Ati-CB1–WT ( n = 6–11) and Ati-CB1–KO ( n = 5–11) mice on HFD. Dbh mRNA levels were measured by ddPCR analysis. ( M ) TH protein levels were measured by flow cytometry in CD11b + F4/80 + -sorted ATMs (left) and CD301 + cells (M2 macrophages, right) in WAT from Ati-CB1–WT ( n = 9) and Ati-CB1–KO ( n = 9) mice on HFD. Data are shown as mean ± SEM. * P

    Techniques Used: Activation Assay, Mouse Assay, Activity Assay, Expressing, Flow Cytometry, Cytometry

    72) Product Images from "Landscape of ribosome-engaged transcript isoforms reveals extensive neuronal cell class-specific alternative splicing programs"

    Article Title: Landscape of ribosome-engaged transcript isoforms reveals extensive neuronal cell class-specific alternative splicing programs

    Journal: Nature neuroscience

    doi: 10.1038/s41593-019-0465-5

    Differential splicing factors expression for cell class-specific splicing programs in mature neurons a . b , Cartoon illustrating the design of splicing reporters for Gabrg2 , Grin1 and Kcnq2 for details). c , RT-PCR for splicing reporters overexpressed in HEK293T cells, Neuro2A (N2A) cells and cultured cortical neurons, or in combination with overexpression of several splicing factors (indicated above ) in N2A cells. On the right , schematic representation of reporter exons amplified and cell types in which a given splicing pattern is enriched are indicated. For each sample, three PCR reactions were performed and band intensity was quantified. Representative images are shown. Below , histograms represent the percentage of inclusion ( in brown) or exclusion ( in light and dark gray ) band intensity relative to the sum intensity of all bands. n=2-3 RT-PCRs, single data points and SEM are indicated. Circles represent quantification of exclusion values for all reporters, triangles, only for Kcnq2 , the quantification of the alternative acceptor e14. Top PCR panel : Expression of the splicing reporter for exon 9 of Gabrg2 leads to differential exon inclusion in non-neuronal (HEK293T, excl. e9) versus neuronal (N2A or cortical neurons, 50% excl. e9) cells. Co-expression of Ptbp1 and, to lower extents, Ptbp2 and Slm1 (enriched in VIP) lead to higher exclusion rates, a pattern significantly enriched in VIP neurons. Co-expression of Rbfox1/2/3 slightly reduces exon exclusion rates, consistent with the splicing pattern observed in purifications of Scnn1a. Middle PCR panel : Similar effects of the same splicing factors can be observed for the splicing pattern of exon 4 of Grin1 . Note that the amplification of Grin1 isoform including e4 generates a doublet band. Lower PCR panel : Exon 13 of Kcnq2 splice reporter is preferentially included in N2A cells. Addition of Ptbp1 and Ptbp2 induces the additional alternative acceptor site usage found in VIP neurons. Overall, these experiments indicate a correlation between splicing factor expression and alternative isoform usage. d, Highest or lowest expression levels in different cortical populations for Ptbp1, Rbofox1, Rbfox3 and Slm1 ( Khdrbs2 ) are indicated by arrows.
    Figure Legend Snippet: Differential splicing factors expression for cell class-specific splicing programs in mature neurons a . b , Cartoon illustrating the design of splicing reporters for Gabrg2 , Grin1 and Kcnq2 for details). c , RT-PCR for splicing reporters overexpressed in HEK293T cells, Neuro2A (N2A) cells and cultured cortical neurons, or in combination with overexpression of several splicing factors (indicated above ) in N2A cells. On the right , schematic representation of reporter exons amplified and cell types in which a given splicing pattern is enriched are indicated. For each sample, three PCR reactions were performed and band intensity was quantified. Representative images are shown. Below , histograms represent the percentage of inclusion ( in brown) or exclusion ( in light and dark gray ) band intensity relative to the sum intensity of all bands. n=2-3 RT-PCRs, single data points and SEM are indicated. Circles represent quantification of exclusion values for all reporters, triangles, only for Kcnq2 , the quantification of the alternative acceptor e14. Top PCR panel : Expression of the splicing reporter for exon 9 of Gabrg2 leads to differential exon inclusion in non-neuronal (HEK293T, excl. e9) versus neuronal (N2A or cortical neurons, 50% excl. e9) cells. Co-expression of Ptbp1 and, to lower extents, Ptbp2 and Slm1 (enriched in VIP) lead to higher exclusion rates, a pattern significantly enriched in VIP neurons. Co-expression of Rbfox1/2/3 slightly reduces exon exclusion rates, consistent with the splicing pattern observed in purifications of Scnn1a. Middle PCR panel : Similar effects of the same splicing factors can be observed for the splicing pattern of exon 4 of Grin1 . Note that the amplification of Grin1 isoform including e4 generates a doublet band. Lower PCR panel : Exon 13 of Kcnq2 splice reporter is preferentially included in N2A cells. Addition of Ptbp1 and Ptbp2 induces the additional alternative acceptor site usage found in VIP neurons. Overall, these experiments indicate a correlation between splicing factor expression and alternative isoform usage. d, Highest or lowest expression levels in different cortical populations for Ptbp1, Rbofox1, Rbfox3 and Slm1 ( Khdrbs2 ) are indicated by arrows.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Over Expression, Amplification, Polymerase Chain Reaction

    73) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    74) Product Images from "Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females"

    Article Title: Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092841

    Expression of MPF components and its activity is increased in the oocytes from aged females. ( a ) RT-PCR quantification of mRNA coding for CDK1 and B-type cyclins, as well as loading control Gapdh in the GV oocytes (0 h) from different age groups. For quantification of total RNA content in oocytes from YF and AF groups see Figure S3a . Values obtained for the YF group were set as 100%. Data was derived from at least four experiments of biologically different samples. Columns represent mean; error bars ± SD; ns non-significant; * p
    Figure Legend Snippet: Expression of MPF components and its activity is increased in the oocytes from aged females. ( a ) RT-PCR quantification of mRNA coding for CDK1 and B-type cyclins, as well as loading control Gapdh in the GV oocytes (0 h) from different age groups. For quantification of total RNA content in oocytes from YF and AF groups see Figure S3a . Values obtained for the YF group were set as 100%. Data was derived from at least four experiments of biologically different samples. Columns represent mean; error bars ± SD; ns non-significant; * p

    Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    75) Product Images from "Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging"

    Article Title: Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00065

    Expression of inflammatory, phagocytic and metabolism genes in LFD and HFD microglia. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS. Microglia were FACS isolated and RNA was extracted and quantified using RT-qPCR. RNA expression levels were normalized to Hmbs levels as in internal control and the expression levels in PBS-injected LFD mice were set at 1. Gene expression levels were compared between both PBS- and LPS-injected mice and between HFD and LFD animals. (A) proinflammatory cytokines ( Il-1 β, Il-6 , and Tnf -α), (B) immune response ( Spp1 and Cybb ), (C) phagocytosis ( Axl, Lgals3 ), and (D) metabolism ( Apoe ) genes were significantly upregulated after LPS injection, but no significant difference between HFD+LPS and LFD+LPS samples was detected. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p
    Figure Legend Snippet: Expression of inflammatory, phagocytic and metabolism genes in LFD and HFD microglia. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS. Microglia were FACS isolated and RNA was extracted and quantified using RT-qPCR. RNA expression levels were normalized to Hmbs levels as in internal control and the expression levels in PBS-injected LFD mice were set at 1. Gene expression levels were compared between both PBS- and LPS-injected mice and between HFD and LFD animals. (A) proinflammatory cytokines ( Il-1 β, Il-6 , and Tnf -α), (B) immune response ( Spp1 and Cybb ), (C) phagocytosis ( Axl, Lgals3 ), and (D) metabolism ( Apoe ) genes were significantly upregulated after LPS injection, but no significant difference between HFD+LPS and LFD+LPS samples was detected. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p

    Techniques Used: Expressing, Injection, FACS, Isolation, Quantitative RT-PCR, RNA Expression, Mouse Assay

    Expression of immune-related genes in the hypothalamus of PBS/LPS-treated LFD and HFD mice. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS, total RNA was extracted from the hypothalamus and analyzed using RT-qPCR. The gene expression of (A) proinflammatory cytokines ( Il-1β and Tnf-α ), (B) genes relating to immune response ( CD44, Cryab, Sirpa, Spp1, Ifitm3 , and Ifitm2 ), (C) phagocytic markers ( Axl, Lgals3, CD36 , and Clec7a ), and (D) genes relating to lipid metabolism ( Apoe, Csf-1, Lpl , and Lrp12 ) were compared between HFD and LFD animals, but also between samples with or without LPS injection. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p
    Figure Legend Snippet: Expression of immune-related genes in the hypothalamus of PBS/LPS-treated LFD and HFD mice. 6-month-old HFD and LFD animals were i.p. injected with LPS or PBS, total RNA was extracted from the hypothalamus and analyzed using RT-qPCR. The gene expression of (A) proinflammatory cytokines ( Il-1β and Tnf-α ), (B) genes relating to immune response ( CD44, Cryab, Sirpa, Spp1, Ifitm3 , and Ifitm2 ), (C) phagocytic markers ( Axl, Lgals3, CD36 , and Clec7a ), and (D) genes relating to lipid metabolism ( Apoe, Csf-1, Lpl , and Lrp12 ) were compared between HFD and LFD animals, but also between samples with or without LPS injection. Open circles depict LFD and closed circles depict HFD samples ( n = 5 mice, mean ± SEM is depicted, Student’s t -test, ∗ p

    Techniques Used: Expressing, Mouse Assay, Injection, Quantitative RT-PCR

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: .. Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Q-PCR was performed on an ABI PRISM 7200 Sequence Detector System using SYBR Green PCR Master Mix (Applied Biosystems); the specific primers are listed in Supporting Information Table 4.

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability
    Article Snippet: .. Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen). .. Concentration and integrity of the RNA extracted from FAC sorted cells were determined with an Agilent 2100 Bioanalyzer and by use of the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For quantitative PCR, PCR amplifications were performed using SYBR Green PCR Master Mix (ABI) and analyzed by the ABI 7500 sequence detection system.

    Amplification:

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability
    Article Snippet: Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen). .. The extracted RNA was treated with Amplification Grade DNAse (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA).

    Article Title: The Human Heart Contains Distinct Macrophage Subsets with Divergent Origins and Functions
    Article Snippet: .. cDNA amplification and RT-PCR RNA was extracted from the cardiac slices or cultured cells using the RNeasy RNA micro kit (Qiagen). ..

    Nucleic Acid Electrophoresis:

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Synthesized:

    Article Title: The Human Heart Contains Distinct Macrophage Subsets with Divergent Origins and Functions
    Article Snippet: cDNA amplification and RT-PCR RNA was extracted from the cardiac slices or cultured cells using the RNeasy RNA micro kit (Qiagen). .. For cultured macrophages, cDNA was synthesized using the iScript™ Reverse Transcription Supermix (Bio-Rad) and pre-amplified using the Sso Advanced PreAmp Supermix kit (Bio-Rad).

    Isolation:

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease
    Article Snippet: Once the microvesicles were isolated and digested with nucleases, the pellet was processed by the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. .. The RNeasy Plus Micro kit (Qiagen) is designed to remove genomic DNA and was used according to the manufacturer’s instructions and eluted in 16 µl nuclease-free water.

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease
    Article Snippet: .. For small RNA isolation using the RNeasy Micro kit or RNeasy Plus Micro kit, the miRNA isolation method was followed according to the manufacturer’s instructions. .. RNA was isolated from whole urine using the ZR urine RNA isolation kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions.

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: .. Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Q-PCR was performed on an ABI PRISM 7200 Sequence Detector System using SYBR Green PCR Master Mix (Applied Biosystems); the specific primers are listed in Supporting Information Table 4.

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability
    Article Snippet: .. Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen). .. Concentration and integrity of the RNA extracted from FAC sorted cells were determined with an Agilent 2100 Bioanalyzer and by use of the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen. .. Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells). .. In both cases, only longer RNA fragments ( > 200 nucleotides) are purified, although the workflow can be modified to isolate small RNAs such as microRNAs, 5,8S rRNA, 5S RNA, tRNA’s,… (Additional file : Figure S1).

    Article Title: Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis
    Article Snippet: .. Total RNA was extracted from isolated crypts using an RNeasy Plus Micro Extraction Kit (Qiagen, Hilden, Germany). .. An expression analysis was performed using the RT2 Profiler PCR Array (Mouse Cell Junction Pathway Finder).

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells. .. The RNA purification protocol is fast (only 20–30 min), easy and consistently delivers high-quality RNA samples.

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: .. Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Next-Generation Sequencing:

    Article Title: Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice
    Article Snippet: Paragraph title: Next generation sequencing ... Cells from embryos with the same genotype were pooled for RNA preperation with RNeasy Plus micro kit (Qiagen 74034).

    Sequencing:

    Article Title: Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice
    Article Snippet: Cells from embryos with the same genotype were pooled for RNA preperation with RNeasy Plus micro kit (Qiagen 74034). .. RNA samples with RNA integrity number > 8.0 were shipped to Beijin Genome Institute for a library preparation and sequencing (Illumina HiSeq 2500).

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Q-PCR was performed on an ABI PRISM 7200 Sequence Detector System using SYBR Green PCR Master Mix (Applied Biosystems); the specific primers are listed in Supporting Information Table 4.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: In addition, we incorporated an in depth quality assessment that could serve as an example for other sequencing experiments and experimental set ups. .. We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen.

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For quantitative PCR, PCR amplifications were performed using SYBR Green PCR Master Mix (ABI) and analyzed by the ABI 7500 sequence detection system.

    Quantitative RT-PCR:

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: .. Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Purification:

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen. .. The RNA purification protocol is fast (only 20–30 min), easy and consistently delivers high-quality RNA samples.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells). .. In both cases, only longer RNA fragments ( > 200 nucleotides) are purified, although the workflow can be modified to isolate small RNAs such as microRNAs, 5,8S rRNA, 5S RNA, tRNA’s,… (Additional file : Figure S1).

    SYBR Green Assay:

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Q-PCR was performed on an ABI PRISM 7200 Sequence Detector System using SYBR Green PCR Master Mix (Applied Biosystems); the specific primers are listed in Supporting Information Table 4.

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For quantitative PCR, PCR amplifications were performed using SYBR Green PCR Master Mix (ABI) and analyzed by the ABI 7500 sequence detection system.

    Concentration Assay:

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability
    Article Snippet: Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen). .. Concentration and integrity of the RNA extracted from FAC sorted cells were determined with an Agilent 2100 Bioanalyzer and by use of the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).

    Transgenic Assay:

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells). .. Ten replicates of 20,000 sorted cells from the Tg (fli1a :EGFP ) transgenic zebrafish line were used as input for comparative RNA-isolation between the two kits.

    Polymerase Chain Reaction:

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Q-PCR was performed on an ABI PRISM 7200 Sequence Detector System using SYBR Green PCR Master Mix (Applied Biosystems); the specific primers are listed in Supporting Information Table 4.

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability
    Article Snippet: Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen). .. The UCAMC Molecular Discovery Core performed the PCR analysis.

    Article Title: Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis
    Article Snippet: Total RNA was extracted from isolated crypts using an RNeasy Plus Micro Extraction Kit (Qiagen, Hilden, Germany). .. An expression analysis was performed using the RT2 Profiler PCR Array (Mouse Cell Junction Pathway Finder).

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Cell Culture:

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: .. Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Article Title: The Human Heart Contains Distinct Macrophage Subsets with Divergent Origins and Functions
    Article Snippet: .. cDNA amplification and RT-PCR RNA was extracted from the cardiac slices or cultured cells using the RNeasy RNA micro kit (Qiagen). ..

    Expressing:

    Article Title: Vascular-derived TGF-? increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
    Article Snippet: Quantitative PCR analysis Total RNA was extracted from the sorted cells or total SVZ using Micro RNeasy plus isolation kits (Qiagen) and then reverse-transcribed using a high-capacity reverse transcription kit (Applied Biosystems). .. Each sample was normalized to endogenous Gapdh expression.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: As RNA degradation does not occur at the same rate or to the same extent for every transcript, RNA integrity impacts expression studies highlighting the importance of RNA quality assessment. .. We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen.

    Article Title: Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis
    Article Snippet: Paragraph title: Expression analyses ... Total RNA was extracted from isolated crypts using an RNeasy Plus Micro Extraction Kit (Qiagen, Hilden, Germany).

    FACS:

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen. .. Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: Paragraph title: The RNAqueous micro kit and RNeasy plus micro kit enable high quality RNA purification from low numbers of FACS sorted EGFP positive zebrafish cells ... The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells).

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: .. Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells. .. The RNA purification protocol is fast (only 20–30 min), easy and consistently delivers high-quality RNA samples.

    Modification:

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells). .. In both cases, only longer RNA fragments ( > 200 nucleotides) are purified, although the workflow can be modified to isolate small RNAs such as microRNAs, 5,8S rRNA, 5S RNA, tRNA’s,… (Additional file : Figure S1).

    Lysis:

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: We compared the performance of two RNA isolation kits for obtaining high-quality RNA from FACS sorted cells, i.e. the RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen. .. In addition, sorting into the lysis buffer will protect RNA up until the moment of purification, making it possible to postpone RNA isolation after sorting.

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes
    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells). .. Each set of 20,000 cells was directly sorted into the lysis buffer of the RNA isolation kit (see further).

    other:

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease
    Article Snippet: To remove genomic DNA from the Zymo processed sample, the eluted RNA was resuspended in 350 µl RLT buffer and processed using the RNeasy Plus Micro kit and eluted in 16 µl nuclease-free water.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
    Article Snippet: Semi-quantitative and quantitative RT-PCR Total RNA from mouse adult tissue samples and cultured and isolated cell samples were extracted using RNeasy mini and micro kit (QIAGEN), respectively, according to the manufacturer’s instruction and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). .. For semi-quantitative RT-PCR, PCR amplifications were performed using Taq DNA polymerase and visualized by gel electrophoresis.

    Article Title: The Human Heart Contains Distinct Macrophage Subsets with Divergent Origins and Functions
    Article Snippet: .. cDNA amplification and RT-PCR RNA was extracted from the cardiac slices or cultured cells using the RNeasy RNA micro kit (Qiagen). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen rna column based isolation kits
    Islet1 knock-down affects sensory neuron differentiation. (A-C) In control embryos (A, B) , Rohon-Beard cells (RBs) express runx3 . Islet1 knock-down leads to fewer cells with robust expression of runx3 (C). The asterisk denotes a cell expressing the gene. (D-F’) <t>Tg(-3.4neurog1:gfp)sb4</t> control (D-E’) and E3 morphant (F, F’) 24 hours post-fertilization (hpf) embryos were examined for expression of olig4 (red). (D-E’) In lateral views of the dorsal spinal cord, <t>RNA</t> in situ hybridization reveals expression of the interneuron marker olig4 (red). Green fluorescent protein (GFP) + neurons do not express olig4 and comprise RBs and dorsal lateral ascending interneurons (see Figure 2 ). (F-F’) Islet1 knock-down leads to an increase in the number of olig4 expressing cells within the dorsal spinal cord. However, similar to RBs of control embryos (D-E’) , RB-like neurons do not express detectable levels of olig4 (F) . (G-I) At 72 hpf, dorsal root ganglia (DRGs) are easily identified as GFP + neurons with large somata located near the spinal cord/notochord border. (G, H) In control embryos, DRG neurons project from their soma bipolar axons that extend dorsally and ventrally (asterisks). (I) Islet1 knock-down reduces the number of GFP + DRGs. Furthermore, for the few GFP + DRGs remaining, their axons show abnormal morphologies. Scale bars = 50 μm in A (for A-C ), D (for D-F ’) and G (for G-I ). CtlMO, 5-base mismatched; islet1 (Sp)E3MO; E3MO, E3 morpholino; Uninj, uninjected.
    Rna Column Based Isolation Kits, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna column based isolation kits/product/Qiagen
    Average 99 stars, based on 85 article reviews
    Price from $9.99 to $1999.99
    rna column based isolation kits - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Islet1 knock-down affects sensory neuron differentiation. (A-C) In control embryos (A, B) , Rohon-Beard cells (RBs) express runx3 . Islet1 knock-down leads to fewer cells with robust expression of runx3 (C). The asterisk denotes a cell expressing the gene. (D-F’) Tg(-3.4neurog1:gfp)sb4 control (D-E’) and E3 morphant (F, F’) 24 hours post-fertilization (hpf) embryos were examined for expression of olig4 (red). (D-E’) In lateral views of the dorsal spinal cord, RNA in situ hybridization reveals expression of the interneuron marker olig4 (red). Green fluorescent protein (GFP) + neurons do not express olig4 and comprise RBs and dorsal lateral ascending interneurons (see Figure 2 ). (F-F’) Islet1 knock-down leads to an increase in the number of olig4 expressing cells within the dorsal spinal cord. However, similar to RBs of control embryos (D-E’) , RB-like neurons do not express detectable levels of olig4 (F) . (G-I) At 72 hpf, dorsal root ganglia (DRGs) are easily identified as GFP + neurons with large somata located near the spinal cord/notochord border. (G, H) In control embryos, DRG neurons project from their soma bipolar axons that extend dorsally and ventrally (asterisks). (I) Islet1 knock-down reduces the number of GFP + DRGs. Furthermore, for the few GFP + DRGs remaining, their axons show abnormal morphologies. Scale bars = 50 μm in A (for A-C ), D (for D-F ’) and G (for G-I ). CtlMO, 5-base mismatched; islet1 (Sp)E3MO; E3MO, E3 morpholino; Uninj, uninjected.

    Journal: Neural Development

    Article Title: Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability

    doi: 10.1186/1749-8104-9-19

    Figure Lengend Snippet: Islet1 knock-down affects sensory neuron differentiation. (A-C) In control embryos (A, B) , Rohon-Beard cells (RBs) express runx3 . Islet1 knock-down leads to fewer cells with robust expression of runx3 (C). The asterisk denotes a cell expressing the gene. (D-F’) Tg(-3.4neurog1:gfp)sb4 control (D-E’) and E3 morphant (F, F’) 24 hours post-fertilization (hpf) embryos were examined for expression of olig4 (red). (D-E’) In lateral views of the dorsal spinal cord, RNA in situ hybridization reveals expression of the interneuron marker olig4 (red). Green fluorescent protein (GFP) + neurons do not express olig4 and comprise RBs and dorsal lateral ascending interneurons (see Figure 2 ). (F-F’) Islet1 knock-down leads to an increase in the number of olig4 expressing cells within the dorsal spinal cord. However, similar to RBs of control embryos (D-E’) , RB-like neurons do not express detectable levels of olig4 (F) . (G-I) At 72 hpf, dorsal root ganglia (DRGs) are easily identified as GFP + neurons with large somata located near the spinal cord/notochord border. (G, H) In control embryos, DRG neurons project from their soma bipolar axons that extend dorsally and ventrally (asterisks). (I) Islet1 knock-down reduces the number of GFP + DRGs. Furthermore, for the few GFP + DRGs remaining, their axons show abnormal morphologies. Scale bars = 50 μm in A (for A-C ), D (for D-F ’) and G (for G-I ). CtlMO, 5-base mismatched; islet1 (Sp)E3MO; E3MO, E3 morpholino; Uninj, uninjected.

    Article Snippet: Real time quantitative PCR Total RNA was extracted from FAC sorted GFP+ cells using RNA column-based isolation kits, RNeasy® Micro kit (Qiagen).

    Techniques: Expressing, RNA In Situ Hybridization, Marker

    Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Article Snippet: Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells.

    Techniques: Lysis, Isolation

    Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Article Snippet: Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells.

    Techniques: Lysis, Isolation, FACS

    Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Article Snippet: Based on different quality parameters and comparison of various experimental set-ups, we recommend using the RNeasy Plus Micro Kit for RNA isolation of FACS sorted zebrafish cells.

    Techniques: Purification, FACS, Quantitative RT-PCR, Isolation

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Journal: Kidney international

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    doi: 10.1038/ki.2010.106

    Figure Lengend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Article Snippet: The RNeasy Plus Micro kit (Qiagen) is designed to remove genomic DNA and was used according to the manufacturer’s instructions and eluted in 16 µl nuclease-free water.

    Techniques: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Journal: Kidney international

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    doi: 10.1038/ki.2010.106

    Figure Lengend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Article Snippet: The RNeasy Plus Micro kit (Qiagen) is designed to remove genomic DNA and was used according to the manufacturer’s instructions and eluted in 16 µl nuclease-free water.

    Techniques: Lysis, Isolation, RNA Extraction, Incubation