gfp negative facs  (Qiagen)

 
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    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
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    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 92 stars, based on 38158 article reviews
    Price from $9.99 to $1999.99
    gfp negative facs - by Bioz Stars, 2020-09
    92/100 stars

    Images

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

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

    Journal: eLife

    doi: 10.7554/eLife.36187

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

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

    2) Product Images from "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

    3) Product Images from "Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells"

    Article Title: Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25921-8

    Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. ( A ) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). ( B ) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P
    Figure Legend Snippet: Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. ( A ) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). ( B ) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P

    Techniques Used: Expressing, Over Expression, RNA Sequencing Assay, Plasmid Preparation

    Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. ( A ) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. ( B ) Genes plotted in ( A ) were broken down according to their relative expression in eNSCs and aNSCs as indicated.
    Figure Legend Snippet: Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. ( A ) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. ( B ) Genes plotted in ( A ) were broken down according to their relative expression in eNSCs and aNSCs as indicated.

    Techniques Used: Expressing, Over Expression, RNA Sequencing Assay

    4) 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

    5) 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

    6) 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

    7) Product Images from "Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease"

    Article Title: Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease

    Journal: bioRxiv

    doi: 10.1101/531202

    Dedifferentiation in PSEN1 M146L hiPSC-derived neurons is caused by changes in histone methylation leading to modulation of chromatin accessibility A Differentially methylated histone regions in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ChIP-Seq for H3K4Me3 or H3K27Me3. ( n = 1) B-C Homer TF motif enrichment of all regions with a significant increase or decrease in B H3K4Me3 or C H3K27Me3 (O/S/N = OCT4-SOX2-Nanog). D-E Metascape functional enrichment of all regions with a significant increase or decrease in D H3K4Me3 or E H3K27Me3. F DEGs with a directionally corresponding increase or decrease in H3K4Me3 or H3K27Me3. G Enrichr enrichment of DEGs with significant change in H3K4Me3 and H3K27Me3 using the ENCODE/ChEA Consensus TF database. H ChIP-Seq read profiles around the summits of differential H3K4Me3 or H3K27Me3 methylation for DEGs with significant change in H3K4Me3 or H3K27Me3. I Metascape enrichment of DEGs with significant change in H3K4Me3 or H3K27Me3. J log 2 fold change in chromatin accessibility and RNA expression in genes with (left) increased gene expression and corresponding change in H3K trimethylation state or (right) decreased gene expression and corresponding change in H3K trimethylation state. K Metascape enrichment of genes with differential H3K trimethylation, chromatin accessibility, and gene expression. L-N ATAC-Seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of L genes with increased expression, M genes with decreased expression, or N miRNAs with decreased expression. O Heatmap of log 2 fold change in RNA expression (RNA), chromatin accessibility within the promoter (ATAC), H3K4Me3 status, or H3K27Me3 (projected in negative log 2 space to correspond to expression) status for endotype-associated or previously identified AD-associated genes.
    Figure Legend Snippet: Dedifferentiation in PSEN1 M146L hiPSC-derived neurons is caused by changes in histone methylation leading to modulation of chromatin accessibility A Differentially methylated histone regions in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ChIP-Seq for H3K4Me3 or H3K27Me3. ( n = 1) B-C Homer TF motif enrichment of all regions with a significant increase or decrease in B H3K4Me3 or C H3K27Me3 (O/S/N = OCT4-SOX2-Nanog). D-E Metascape functional enrichment of all regions with a significant increase or decrease in D H3K4Me3 or E H3K27Me3. F DEGs with a directionally corresponding increase or decrease in H3K4Me3 or H3K27Me3. G Enrichr enrichment of DEGs with significant change in H3K4Me3 and H3K27Me3 using the ENCODE/ChEA Consensus TF database. H ChIP-Seq read profiles around the summits of differential H3K4Me3 or H3K27Me3 methylation for DEGs with significant change in H3K4Me3 or H3K27Me3. I Metascape enrichment of DEGs with significant change in H3K4Me3 or H3K27Me3. J log 2 fold change in chromatin accessibility and RNA expression in genes with (left) increased gene expression and corresponding change in H3K trimethylation state or (right) decreased gene expression and corresponding change in H3K trimethylation state. K Metascape enrichment of genes with differential H3K trimethylation, chromatin accessibility, and gene expression. L-N ATAC-Seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of L genes with increased expression, M genes with decreased expression, or N miRNAs with decreased expression. O Heatmap of log 2 fold change in RNA expression (RNA), chromatin accessibility within the promoter (ATAC), H3K4Me3 status, or H3K27Me3 (projected in negative log 2 space to correspond to expression) status for endotype-associated or previously identified AD-associated genes.

    Techniques Used: Derivative Assay, Methylation, Chromatin Immunoprecipitation, Functional Assay, RNA Expression, Expressing

    Modulation of chromatin accessibility drives differential gene expression and dedifferentiation in PSEN1 M146L hiPSC-derived neurons A Differentially accessible regions of chromatin in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ATAC-Seq; top, all differentially accessible regions; bottom, differentially expressed genes with differentially accessible regions of chromatin. Colors maintained A-C. ( n = 2) B MA Plot of differential chromatin accessibility occurring within the promotor region or outside the promotor region. C Violin plot of RNA log 2 fold-change for genes with differential accessibility and gene expression. D Homer TF motif enrichment of all regions with increased accessibility (top, purple) or decreased accessibility (green, bottom). E TF motifs with a significant (adj. p-value
    Figure Legend Snippet: Modulation of chromatin accessibility drives differential gene expression and dedifferentiation in PSEN1 M146L hiPSC-derived neurons A Differentially accessible regions of chromatin in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ATAC-Seq; top, all differentially accessible regions; bottom, differentially expressed genes with differentially accessible regions of chromatin. Colors maintained A-C. ( n = 2) B MA Plot of differential chromatin accessibility occurring within the promotor region or outside the promotor region. C Violin plot of RNA log 2 fold-change for genes with differential accessibility and gene expression. D Homer TF motif enrichment of all regions with increased accessibility (top, purple) or decreased accessibility (green, bottom). E TF motifs with a significant (adj. p-value

    Techniques Used: Expressing, Derivative Assay

    8) Product Images from "Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization"

    Article Title: Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2020.05.004

    Endothelial Lactate Controls Macrophage Polarization and Function upon Muscle Ischemia (A) Scheme illustrating experimental set-up. (B) Gene profiling of unstimulated BMDMs (vehicle) or BMDMs stimulated with mECs wt -CM, mECs Δpfkfb3 -CM. (C and D) Gene expression analysis of arg1 (C) and mrc1 (D) in BMDMs stimulated with fractionated mECs wt -CM and mECs Δpfkfb3 -CM, supplemented with lactate (5 mM) where indicated. (E) Lactate concentration in mECs wt -CM and mECs Δpfkfb3 -CM. (F) Representative images of immunostainings of F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECs wt -CM, mECs Δpfkfb3 -CM with or without lactate supplementation and quantification of CD206 MFI (n = 3). (G) OCR upon injection of oligomycin (oligo), FCCP, and rotenone plus antimycin A after mECs wt -CM, mECs Δpfkfb3 -CM, mECs wt+lac -CM, and mECs Δpfkfb3+lac -CM stimulation (n = 4–5). (H) RNaseq data showing OXPHOS gene expression in muscle macrophages 3 days after HLI (n = 3). (I and J) Representative images (I) and quantification (J) of proliferating MPCs upon incubation with macrophage-derived CM, measured as percentage of EdU + nuclei (red, EdU + ; blue, hoechst). (K and L) MPC fusion analysis: representative images of immunofluorescent DESMIN staining (K) (red, DESMIN; blue, hoechst) and quantification of the number of nuclei per DESMIN + myotube (L). (M) Vegf gene expression in CD45 + cells sorted from pfkfb3 WT and pfkfb3 ΔEC muscle at 3 d. (N) VEGF secretion by BMDMs after stimulation with mECs wt -CM and mECs Δpfkfb3 -CM with or without lactate supplementation. Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (E) and (M). One-way ANOVA with Tukey’s multiple comparisons test in (B). Two-way ANOVA with Tukey’s multiple comparisons test in (C), (D), (G), (J), (L), and (N) ( ∗ p
    Figure Legend Snippet: Endothelial Lactate Controls Macrophage Polarization and Function upon Muscle Ischemia (A) Scheme illustrating experimental set-up. (B) Gene profiling of unstimulated BMDMs (vehicle) or BMDMs stimulated with mECs wt -CM, mECs Δpfkfb3 -CM. (C and D) Gene expression analysis of arg1 (C) and mrc1 (D) in BMDMs stimulated with fractionated mECs wt -CM and mECs Δpfkfb3 -CM, supplemented with lactate (5 mM) where indicated. (E) Lactate concentration in mECs wt -CM and mECs Δpfkfb3 -CM. (F) Representative images of immunostainings of F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECs wt -CM, mECs Δpfkfb3 -CM with or without lactate supplementation and quantification of CD206 MFI (n = 3). (G) OCR upon injection of oligomycin (oligo), FCCP, and rotenone plus antimycin A after mECs wt -CM, mECs Δpfkfb3 -CM, mECs wt+lac -CM, and mECs Δpfkfb3+lac -CM stimulation (n = 4–5). (H) RNaseq data showing OXPHOS gene expression in muscle macrophages 3 days after HLI (n = 3). (I and J) Representative images (I) and quantification (J) of proliferating MPCs upon incubation with macrophage-derived CM, measured as percentage of EdU + nuclei (red, EdU + ; blue, hoechst). (K and L) MPC fusion analysis: representative images of immunofluorescent DESMIN staining (K) (red, DESMIN; blue, hoechst) and quantification of the number of nuclei per DESMIN + myotube (L). (M) Vegf gene expression in CD45 + cells sorted from pfkfb3 WT and pfkfb3 ΔEC muscle at 3 d. (N) VEGF secretion by BMDMs after stimulation with mECs wt -CM and mECs Δpfkfb3 -CM with or without lactate supplementation. Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (E) and (M). One-way ANOVA with Tukey’s multiple comparisons test in (B). Two-way ANOVA with Tukey’s multiple comparisons test in (C), (D), (G), (J), (L), and (N) ( ∗ p

    Techniques Used: Expressing, Concentration Assay, Injection, Incubation, Derivative Assay, Staining, Two Tailed Test

    Lactate-Induced Macrophage Polarization Is MCT1 Dependent (A) Lactate uptake in macrophages isolated from muscle 3 days after HLI. (B) Lactate uptake in BMDMs after stimulation with mECs-CM. mECs-CM was supplemented with vehicle (ctrl), lactate, or MCT1 inhibitor AZD3965 (AZD) (mECs wt+AZD -CM, mECs Δpfkfb3+AZD -CM). (C) Lactate oxidation in BMDMs upon stimulation with mECs wt -CM, mECs Δpfkfb3 -CM, mECs wt+lac -CM, mECs Δpfkfb3+lac -CM, mECs wt+AZD -CM, and mECs Δpfkfb3+AZD -CM. (D) Representative images of immunostainings for F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECs wt+AZD -CM or mECs Δpfkfb3+AZD -CM and flow cytometry quantification of CD206 MFI. (E and F) Representative images (red, EdU + ; blue, hoechst) (E) and quantification (F) of EdU + MPCs. (G and H) Representative DESMIN staining (red, DESMIN; blue, hoechst) (G) and fusion analysis (H) upon stimulation with mECs-CM → BMDMs-CM. (I) VEGF levels in mECs-CM → BMDMs-CM. Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (A) ( ∗ p
    Figure Legend Snippet: Lactate-Induced Macrophage Polarization Is MCT1 Dependent (A) Lactate uptake in macrophages isolated from muscle 3 days after HLI. (B) Lactate uptake in BMDMs after stimulation with mECs-CM. mECs-CM was supplemented with vehicle (ctrl), lactate, or MCT1 inhibitor AZD3965 (AZD) (mECs wt+AZD -CM, mECs Δpfkfb3+AZD -CM). (C) Lactate oxidation in BMDMs upon stimulation with mECs wt -CM, mECs Δpfkfb3 -CM, mECs wt+lac -CM, mECs Δpfkfb3+lac -CM, mECs wt+AZD -CM, and mECs Δpfkfb3+AZD -CM. (D) Representative images of immunostainings for F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECs wt+AZD -CM or mECs Δpfkfb3+AZD -CM and flow cytometry quantification of CD206 MFI. (E and F) Representative images (red, EdU + ; blue, hoechst) (E) and quantification (F) of EdU + MPCs. (G and H) Representative DESMIN staining (red, DESMIN; blue, hoechst) (G) and fusion analysis (H) upon stimulation with mECs-CM → BMDMs-CM. (I) VEGF levels in mECs-CM → BMDMs-CM. Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (A) ( ∗ p

    Techniques Used: Isolation, Flow Cytometry, Staining, Two Tailed Test

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) Product Images from "Developmental hourglass and heterochronic shifts in fin and limb development"

    Article Title: Developmental hourglass and heterochronic shifts in fin and limb development

    Journal: bioRxiv

    doi: 10.1101/2020.01.10.901173

    Transcriptome analysis and orthology assignment. a , The skeletal patterns of a mouse limb (top) and a bamboo shark pectoral fin (bottom). Anterior is to the top; distal is to the right. b , Mouse forelimb buds and bamboo shark pectoral fin buds that were analyzed by RNA-seq. c , Comparison of the accuracy of three orthology assignment methods. Vertical axis, the percentages of correctly assigned Hoxa and Hoxd paralogs (black bars) and Fgf paralogs (white bars). d , Heat map visualization of the transcription profile of Hoxa and Hoxd genes in mouse limb buds (left) and bamboo shark fin buds (right) with scaled TPMs.
    Figure Legend Snippet: Transcriptome analysis and orthology assignment. a , The skeletal patterns of a mouse limb (top) and a bamboo shark pectoral fin (bottom). Anterior is to the top; distal is to the right. b , Mouse forelimb buds and bamboo shark pectoral fin buds that were analyzed by RNA-seq. c , Comparison of the accuracy of three orthology assignment methods. Vertical axis, the percentages of correctly assigned Hoxa and Hoxd paralogs (black bars) and Fgf paralogs (white bars). d , Heat map visualization of the transcription profile of Hoxa and Hoxd genes in mouse limb buds (left) and bamboo shark fin buds (right) with scaled TPMs.

    Techniques Used: RNA Sequencing Assay

    Confirmation analyses of the transcriptome comparison. a , Cross-species comparisons of transcriptome data between the two species with indicated distance methods. Note that these methods consistently show the closest distance around E10.5 and st. 27.5– 30. b , Percentages of stage-associated genes with 1.0 ≥ z-score of mouse limb buds (left) and bamboo shark fin buds (right). Note that both species show a low percentage of stage-associated genes during the middle stages. c , Counts (left) and fractions (right) of tissue-associated genes expressed in mouse limb buds. Tissue specificity was evaluated by entropy using RNA-seq data from 71 mouse tissues. A gene with 0.65 ≤ entropy was considered a tissue-specific gene. In the right panel, gene counts were normalized based on the counts of total expressed genes. Note that the number of tissue-associated genes was lowest at E10.5.
    Figure Legend Snippet: Confirmation analyses of the transcriptome comparison. a , Cross-species comparisons of transcriptome data between the two species with indicated distance methods. Note that these methods consistently show the closest distance around E10.5 and st. 27.5– 30. b , Percentages of stage-associated genes with 1.0 ≥ z-score of mouse limb buds (left) and bamboo shark fin buds (right). Note that both species show a low percentage of stage-associated genes during the middle stages. c , Counts (left) and fractions (right) of tissue-associated genes expressed in mouse limb buds. Tissue specificity was evaluated by entropy using RNA-seq data from 71 mouse tissues. A gene with 0.65 ≤ entropy was considered a tissue-specific gene. In the right panel, gene counts were normalized based on the counts of total expressed genes. Note that the number of tissue-associated genes was lowest at E10.5.

    Techniques Used: RNA Sequencing Assay

    Detection of heterochronic gene expression between mouse limb buds and bamboo shark fin buds. a , Clustering analysis of gene expression dynamics. Each column represents an ortholog pair between the bamboo shark and the mouse. Each row indicates scaled gene expression at a time point indicated to the right of the heat map. Values are scaled TPMs. b, c , Whole-mount in situ hybridization of Hand2 ( b ) and Vcan ( c ) as examples of the heterochronic genes detected in a . Asterisks, background signals; scale bars, 200 μm. d, e , Expression of Shh and related genes in mouse limb buds ( d ) and bamboo shark fin buds ( e ), respectively. The rectangles indicate the expression peaks of Shh, Hoxd9 , and Hoxd10 (red) and Shh target genes (yellow).
    Figure Legend Snippet: Detection of heterochronic gene expression between mouse limb buds and bamboo shark fin buds. a , Clustering analysis of gene expression dynamics. Each column represents an ortholog pair between the bamboo shark and the mouse. Each row indicates scaled gene expression at a time point indicated to the right of the heat map. Values are scaled TPMs. b, c , Whole-mount in situ hybridization of Hand2 ( b ) and Vcan ( c ) as examples of the heterochronic genes detected in a . Asterisks, background signals; scale bars, 200 μm. d, e , Expression of Shh and related genes in mouse limb buds ( d ) and bamboo shark fin buds ( e ), respectively. The rectangles indicate the expression peaks of Shh, Hoxd9 , and Hoxd10 (red) and Shh target genes (yellow).

    Techniques Used: Expressing, In Situ Hybridization

    Hourglass-shaped conservation of the transcriptome profile between fins and limbs. a , Euclidean distances of the transcriptome profiles. Every combination of time points of bamboo shark fin buds and mouse limb buds is shown. The darker colors indicate a greater similarity between gene expression profiles. b , A line plot of the Euclidean distances shown in ( a ). The x axis indicates the mouse limb stages, and the y axis is the Euclidean distance. c , The same as ( a ) except that only Hoxd genes are included. d, e , Scatter plots of the first and second principal components ( d ) and of the second and third components ( e ). Arrows in ( e ) indicate the time-order of transcriptome data. f , Count of tissue-associated genes expressed in mouse forelimb buds. Genes with 0.65 ≤ entropy were counted.
    Figure Legend Snippet: Hourglass-shaped conservation of the transcriptome profile between fins and limbs. a , Euclidean distances of the transcriptome profiles. Every combination of time points of bamboo shark fin buds and mouse limb buds is shown. The darker colors indicate a greater similarity between gene expression profiles. b , A line plot of the Euclidean distances shown in ( a ). The x axis indicates the mouse limb stages, and the y axis is the Euclidean distance. c , The same as ( a ) except that only Hoxd genes are included. d, e , Scatter plots of the first and second principal components ( d ) and of the second and third components ( e ). Arrows in ( e ) indicate the time-order of transcriptome data. f , Count of tissue-associated genes expressed in mouse forelimb buds. Genes with 0.65 ≤ entropy were counted.

    Techniques Used: Expressing

    18) Product Images from "Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease"

    Article Title: Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease

    Journal: bioRxiv

    doi: 10.1101/531202

    A Principal Component Analysis of NDC, PSEN1 M146L , and PSEN1 A246E hiPSC-derived neurons (n = 4). B-C log 2 fold change of differentially expressed genes in B PSEN1 M146L or C PSEN1 A246E hiPSC-derived neurons relative to non-demented control (NDC) with a False Discovery Rate (FDR) adjusted p-value (q-value)
    Figure Legend Snippet: A Principal Component Analysis of NDC, PSEN1 M146L , and PSEN1 A246E hiPSC-derived neurons (n = 4). B-C log 2 fold change of differentially expressed genes in B PSEN1 M146L or C PSEN1 A246E hiPSC-derived neurons relative to non-demented control (NDC) with a False Discovery Rate (FDR) adjusted p-value (q-value)

    Techniques Used: Derivative Assay

    A PCA of NDC and PSEN1 M139T human brain sample microarray expression from Antonell et al. 2013; outlier NDC sample in red. B-C Circos plot of overlapping gene and genes with shared gene ontology between PSEN1 M139T human brain samples and PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons for B upregulated genes and C downregulated genes. D-E Enrichment analysis of differentially expressed genes in PSEN1 M139T human brain samples using the GO – Biological Process and Reactome databases by D Panther or E Metascape enrichment. F Pre-ranked GSEA normalized enrichment scores in PSEN1 M139T human brain samples using the Hallmark database (FWER
    Figure Legend Snippet: A PCA of NDC and PSEN1 M139T human brain sample microarray expression from Antonell et al. 2013; outlier NDC sample in red. B-C Circos plot of overlapping gene and genes with shared gene ontology between PSEN1 M139T human brain samples and PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons for B upregulated genes and C downregulated genes. D-E Enrichment analysis of differentially expressed genes in PSEN1 M139T human brain samples using the GO – Biological Process and Reactome databases by D Panther or E Metascape enrichment. F Pre-ranked GSEA normalized enrichment scores in PSEN1 M139T human brain samples using the Hallmark database (FWER

    Techniques Used: Microarray, Expressing, Derivative Assay

    PSEN1 hiPSC-derived neurons undergo dedifferentiation through the activation and repression of key endotypes A Patient-derived Non-Demented Control (NDC), PSEN1 M146L , and PSEN1 A246E hiPSCs were differentiated into neurons and purified by CD44 − /CD184 − selection. B Overlap of differentially expressed genes (DEGs) in PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons relative to non-demented control (NDC) with a False Discovery Rate (FDR) adjusted p-value (q-value)
    Figure Legend Snippet: PSEN1 hiPSC-derived neurons undergo dedifferentiation through the activation and repression of key endotypes A Patient-derived Non-Demented Control (NDC), PSEN1 M146L , and PSEN1 A246E hiPSCs were differentiated into neurons and purified by CD44 − /CD184 − selection. B Overlap of differentially expressed genes (DEGs) in PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons relative to non-demented control (NDC) with a False Discovery Rate (FDR) adjusted p-value (q-value)

    Techniques Used: Derivative Assay, Activation Assay, Purification, Selection

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

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

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

    Journal: eLife

    doi: 10.7554/eLife.36187

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

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

    33) Product Images from "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

    34) 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

    35) 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

    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 "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

    38) 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

    39) Product Images from "Phf6 loss enhances HSC self-renewal driving tumor initiation and leukemia stem cell activity in T-ALL."

    Article Title: Phf6 loss enhances HSC self-renewal driving tumor initiation and leukemia stem cell activity in T-ALL.

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-18-1005

    Phf6 mutations are early events in T-ALL and loss of Phf6 expands the hematopoietic stem compartment. A, Integrated Sequential Network (ISN) illustrating the sequential order of mutations (nodes) in diagnosis and relapse ALL samples (n = 37) by pooling evolutionary paths (arrows) across patients. B, FACS plots at the top show representative analysis of total myeloid progenitor cells (MyP: Lin − CD117 + Sca1 − ) and total hematopoietic stem and progenitor cells (LSK: Lin − CD117 + Sca1 + ) from Phf6 wild-type ( Phf6 +/Y Vav-iCre ) and Phf6 knockout ( Phf6 −/Y Vav-iCre ) littermates at 8 weeks of age. FACS plots at the bottom show representative analysis of LSK subpopulations: long-term HSCs (LT-HSCs: Lin − CD117 + Sca1 + CD150 + CD48 − ), short-term HSCs (ST-HSCs: Lin − CD117 + Sca1 + CD150 − CD48 − ), MPP2 (Lin − CD117 + Sca1 + CD150 + CD48 + ) and MPP3 (Lin − CD117 + Sca1 + CD150 − CD48 + ). For each representative population, frequencies reflect the mean percentage gated on total live cells. C, Frequency of LSK cells derived from Phf6 wild-type (n = 5) and Phf6 knockout (n = 4) littermates at 8 weeks of age. D, Quantification of total LSK cell numbers of populations depicted in B and C. E, The frequency of LT-HSCs, ST-HSCs, MPP2, MPP3 and lymphoid-restricted MPP4 (Lin − CD117 + Sca1 + CD135 + CD150 − ) progenitors derived from Phf6 wild-type (n = 5) and Phf6 knockout (n = 4) littermates. F, Absolute number of LT-HSCs and ST-HSCs as in B and E. G, Quantification of total cell numbers in multipotent (MPP2, and MPP3) and lymphoid restricted (MPP4) progenitor cell populations as in B and E. H, Total donor-derived cell frequencies in peripheral blood after primary, secondary and tertiary competitive transplantation of bone marrow cells from Phf6 wild-type ( Phf6 +/Y Vav-iCre ) or Phf6 knockout ( Phf6 −/Y Vav-iCre ) control mice (n = 6 - 7 recipients per group). Plots of frequency and absolute cell number show individual mice; bars represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test.
    Figure Legend Snippet: Phf6 mutations are early events in T-ALL and loss of Phf6 expands the hematopoietic stem compartment. A, Integrated Sequential Network (ISN) illustrating the sequential order of mutations (nodes) in diagnosis and relapse ALL samples (n = 37) by pooling evolutionary paths (arrows) across patients. B, FACS plots at the top show representative analysis of total myeloid progenitor cells (MyP: Lin − CD117 + Sca1 − ) and total hematopoietic stem and progenitor cells (LSK: Lin − CD117 + Sca1 + ) from Phf6 wild-type ( Phf6 +/Y Vav-iCre ) and Phf6 knockout ( Phf6 −/Y Vav-iCre ) littermates at 8 weeks of age. FACS plots at the bottom show representative analysis of LSK subpopulations: long-term HSCs (LT-HSCs: Lin − CD117 + Sca1 + CD150 + CD48 − ), short-term HSCs (ST-HSCs: Lin − CD117 + Sca1 + CD150 − CD48 − ), MPP2 (Lin − CD117 + Sca1 + CD150 + CD48 + ) and MPP3 (Lin − CD117 + Sca1 + CD150 − CD48 + ). For each representative population, frequencies reflect the mean percentage gated on total live cells. C, Frequency of LSK cells derived from Phf6 wild-type (n = 5) and Phf6 knockout (n = 4) littermates at 8 weeks of age. D, Quantification of total LSK cell numbers of populations depicted in B and C. E, The frequency of LT-HSCs, ST-HSCs, MPP2, MPP3 and lymphoid-restricted MPP4 (Lin − CD117 + Sca1 + CD135 + CD150 − ) progenitors derived from Phf6 wild-type (n = 5) and Phf6 knockout (n = 4) littermates. F, Absolute number of LT-HSCs and ST-HSCs as in B and E. G, Quantification of total cell numbers in multipotent (MPP2, and MPP3) and lymphoid restricted (MPP4) progenitor cell populations as in B and E. H, Total donor-derived cell frequencies in peripheral blood after primary, secondary and tertiary competitive transplantation of bone marrow cells from Phf6 wild-type ( Phf6 +/Y Vav-iCre ) or Phf6 knockout ( Phf6 −/Y Vav-iCre ) control mice (n = 6 - 7 recipients per group). Plots of frequency and absolute cell number show individual mice; bars represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test.

    Techniques Used: FACS, Knock-Out, Derivative Assay, Transplantation Assay, Mouse Assay, Standard Deviation, Two Tailed Test

    Phf6 deletion in adult LT-HSCs increases hematopoietic repopulation capacity associated with increased chromatin accessibility and transcriptional upregulation of JAK-STAT signature genes. A, Frequency of total donor-derived cells in peripheral blood 5, 10 and 15 weeks after transplantation of 500 LT-HSCs sorted from Phf6 wild-type ( Phf6 f/Y Rosa26 +/+ ) or Phf6 knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) donor mice (n = 10 recipients per group). B, The absolute number of hematopoietic stem and progenitor cells (LT- and ST-HSCs, MPP2-4) and myeloid-restricted progenitor cells (MEP, CMP, GMP) derived from 500 Phf6 wild-type or Phf6 knockout sorted LT-HSCs in the bone marrow of recipient mice16 weeks after transplantations (n = 5 per group), as in A. C, Frequency of total donor-derived reconstitution in peripheral blood after secondary transplantation of 1000 re-sorted Phf6 wild-type or Phf6 knockout LT-HSCs (n = 10 per group). D, Relative distribution of B cell (B220 + ), T cell (CD3 + ) and myeloid (CD11b + ) populations in peripheral blood 15 weeks post-transplant, as in C. Values are shown for individual mice. Graphs represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test. *** indicates P
    Figure Legend Snippet: Phf6 deletion in adult LT-HSCs increases hematopoietic repopulation capacity associated with increased chromatin accessibility and transcriptional upregulation of JAK-STAT signature genes. A, Frequency of total donor-derived cells in peripheral blood 5, 10 and 15 weeks after transplantation of 500 LT-HSCs sorted from Phf6 wild-type ( Phf6 f/Y Rosa26 +/+ ) or Phf6 knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) donor mice (n = 10 recipients per group). B, The absolute number of hematopoietic stem and progenitor cells (LT- and ST-HSCs, MPP2-4) and myeloid-restricted progenitor cells (MEP, CMP, GMP) derived from 500 Phf6 wild-type or Phf6 knockout sorted LT-HSCs in the bone marrow of recipient mice16 weeks after transplantations (n = 5 per group), as in A. C, Frequency of total donor-derived reconstitution in peripheral blood after secondary transplantation of 1000 re-sorted Phf6 wild-type or Phf6 knockout LT-HSCs (n = 10 per group). D, Relative distribution of B cell (B220 + ), T cell (CD3 + ) and myeloid (CD11b + ) populations in peripheral blood 15 weeks post-transplant, as in C. Values are shown for individual mice. Graphs represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test. *** indicates P

    Techniques Used: Derivative Assay, Transplantation Assay, Knock-Out, Mouse Assay, Standard Deviation, Two Tailed Test

    Phf6 deletion in adult hematopoiesis increases hematopoietic stem cell self-renewal following long-term competitive serial transplantation. A, Representative FACS plots of total donor-derived myeloid progenitor cells (MyP: Lin − CD117 + Sca1 − ) and total hematopoietic stem and progenitor cells (LSK: Lin − CD117 + Sca1 + ) 8-10 weeks post transplantation of lineage negative cells isolated from Phf6 wild-type ( Phf6 f/Y Rosa26 +/+ ) or Phf6 knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) mice. For each representative population, frequencies reflect the mean percentage gated on total live cells (n = 5 per group). B, Absolute number of donor-derived LT-HSC, MPP2, MPP3 and MPP4 cells in recipients of Phf6 wild-type or Phf6 knockout donor bone marrow cells. C, Total donor-derived cell reconstitution in peripheral blood after primary, secondary and tertiary competitive transplantation of bone marrow cells from Phf6 wild-type and Phf6 knockout mice (n = 5–7 recipients per group). D, Total number of donor-derived wild-type ( Phf6 f/Y Rosa26 +/+ ) and knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) LSK cells in the bone marrow of recipient mice 24 weeks after primary (n = 6 per group), secondary (n = 5 and 4 respectively) and tertiary (n = 5 per group) competitive mixed bone-marrow transplantation. E, Representative FACS plots of total donor-derived stem and progenitor cells in mice after primary, secondary and tertiary competitive mixed bone marrow transplantation with Phf6 wild-type or Phf6 knockout bone marrow cells. For each population, the mean percentage gated on total LSK cells is indicated. F, Frequency and absolute cell number of LT-HSCs in primary, secondary and tertiary competitive mixed bone marrow recipients as in D and E. G, Frequency of total multipotent progenitor cells (MPP) in primary, secondary and tertiary competitive mixed bone marrow recipients as in D and E. H, Absolute numbers of lymphoid-restricted MPP4 progenitor cells in primary and tertiary competitive mixed bone marrow recipients as in D and E. Plots of both frequency and absolute cell number show individual mice; bars represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test. *** indicates P
    Figure Legend Snippet: Phf6 deletion in adult hematopoiesis increases hematopoietic stem cell self-renewal following long-term competitive serial transplantation. A, Representative FACS plots of total donor-derived myeloid progenitor cells (MyP: Lin − CD117 + Sca1 − ) and total hematopoietic stem and progenitor cells (LSK: Lin − CD117 + Sca1 + ) 8-10 weeks post transplantation of lineage negative cells isolated from Phf6 wild-type ( Phf6 f/Y Rosa26 +/+ ) or Phf6 knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) mice. For each representative population, frequencies reflect the mean percentage gated on total live cells (n = 5 per group). B, Absolute number of donor-derived LT-HSC, MPP2, MPP3 and MPP4 cells in recipients of Phf6 wild-type or Phf6 knockout donor bone marrow cells. C, Total donor-derived cell reconstitution in peripheral blood after primary, secondary and tertiary competitive transplantation of bone marrow cells from Phf6 wild-type and Phf6 knockout mice (n = 5–7 recipients per group). D, Total number of donor-derived wild-type ( Phf6 f/Y Rosa26 +/+ ) and knockout ( Phf6 −/Y Rosa26 +/Cre-ERT2 ) LSK cells in the bone marrow of recipient mice 24 weeks after primary (n = 6 per group), secondary (n = 5 and 4 respectively) and tertiary (n = 5 per group) competitive mixed bone-marrow transplantation. E, Representative FACS plots of total donor-derived stem and progenitor cells in mice after primary, secondary and tertiary competitive mixed bone marrow transplantation with Phf6 wild-type or Phf6 knockout bone marrow cells. For each population, the mean percentage gated on total LSK cells is indicated. F, Frequency and absolute cell number of LT-HSCs in primary, secondary and tertiary competitive mixed bone marrow recipients as in D and E. G, Frequency of total multipotent progenitor cells (MPP) in primary, secondary and tertiary competitive mixed bone marrow recipients as in D and E. H, Absolute numbers of lymphoid-restricted MPP4 progenitor cells in primary and tertiary competitive mixed bone marrow recipients as in D and E. Plots of both frequency and absolute cell number show individual mice; bars represent mean ± standard deviation. P values were calculated using two-tailed Student’s t -test. *** indicates P

    Techniques Used: Transplantation Assay, FACS, Derivative Assay, Isolation, Knock-Out, Mouse Assay, Standard Deviation, Two Tailed Test

    40) Product Images from "Aberrant astrocyte protein secretion contributes to altered neuronal development in diverse disorders"

    Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in diverse disorders

    Journal: bioRxiv

    doi: 10.1101/2020.02.17.939991

    Immunopanned astrocyte and neuron cultures for the study of NDs. a. Schematic of the procedure: P7 mouse cortex is digested with papain to produce a single cell suspension, which undergoes a series of negative selection steps to deplete unwanted cells (endothelia, microglia, oligodendrocytes), followed by positive selection for astrocytes using an antibody against ACSA2. b. qRT-PCR for cell-type markers from mRNA collected from IP astrocytes compared to P7 mouse cortex demonstrates enrichment for astrocytes (Gfap), a depletion of neurons (Syt1), microglia (Cd68), fibroblasts (Fgfr4), and a decrease in oligodendrocyte precursor cells (OPCs; Cspg4) in WT and ND IP astrocyte cultures. N=6 cultures per genotype. c. Immunostaining IP-astrocyte cultures for astrocyte marker GLAST (cyan, Slc1a3) and nuclei (white, DAPI) demonstrates the majority of cells express this protein. d-f. WT ACM supports WT neurite outgrowth, whereas RTT ACM does not. d. Schematic of the immunopanning procedure to isolate cortical neurons: P7 mouse cortex is digested with papain to produce a single cell suspension, which undergoes a series of negative selection steps to deplete unwanted cells (endothelia, microglia), followed by positive selection for neurons using an antibody against NCAM-L1. e-f. Culturing WT cortical neurons for 48 hours with WT astrocyte conditioned media (ACM) increases neurite outgrowth, whereas RTT ACM does not. e. Example images, neurons immunostained with MAP2 and tau, merged image shown. f. Quantification of total neurite outgrowth (dendrite + axon). Example experiment shown, experiment repeated 3 times with same result. Bar graph represents mean ± s.e.m. Number in bar = neurons analyzed. Statistics one-way ANOVA on ranks, p value compared to neurons alone. g. Overview of project workflow. See also Figure S1.
    Figure Legend Snippet: Immunopanned astrocyte and neuron cultures for the study of NDs. a. Schematic of the procedure: P7 mouse cortex is digested with papain to produce a single cell suspension, which undergoes a series of negative selection steps to deplete unwanted cells (endothelia, microglia, oligodendrocytes), followed by positive selection for astrocytes using an antibody against ACSA2. b. qRT-PCR for cell-type markers from mRNA collected from IP astrocytes compared to P7 mouse cortex demonstrates enrichment for astrocytes (Gfap), a depletion of neurons (Syt1), microglia (Cd68), fibroblasts (Fgfr4), and a decrease in oligodendrocyte precursor cells (OPCs; Cspg4) in WT and ND IP astrocyte cultures. N=6 cultures per genotype. c. Immunostaining IP-astrocyte cultures for astrocyte marker GLAST (cyan, Slc1a3) and nuclei (white, DAPI) demonstrates the majority of cells express this protein. d-f. WT ACM supports WT neurite outgrowth, whereas RTT ACM does not. d. Schematic of the immunopanning procedure to isolate cortical neurons: P7 mouse cortex is digested with papain to produce a single cell suspension, which undergoes a series of negative selection steps to deplete unwanted cells (endothelia, microglia), followed by positive selection for neurons using an antibody against NCAM-L1. e-f. Culturing WT cortical neurons for 48 hours with WT astrocyte conditioned media (ACM) increases neurite outgrowth, whereas RTT ACM does not. e. Example images, neurons immunostained with MAP2 and tau, merged image shown. f. Quantification of total neurite outgrowth (dendrite + axon). Example experiment shown, experiment repeated 3 times with same result. Bar graph represents mean ± s.e.m. Number in bar = neurons analyzed. Statistics one-way ANOVA on ranks, p value compared to neurons alone. g. Overview of project workflow. See also Figure S1.

    Techniques Used: Selection, Quantitative RT-PCR, Immunostaining, Marker

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    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.
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    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

    Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. ( A ) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). ( B ) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P

    Journal: Scientific Reports

    Article Title: Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells

    doi: 10.1038/s41598-018-25921-8

    Figure Lengend Snippet: Effect on gene expression of Bmi1 overexpression in eNSCs compared to aNSCs. ( A ) Comparison of transcript levels measured by RNA-seq (reads per kilobase per million reads mapped, RPKM) in embryonic and adult NSCs (empty vector control, average of two biological replicates). ( B ) Gene ontology categories enriched for genes down-regulated (top) or up-regulated (bottom) upon Bmi1 overexpression in eNSCs (red bars) or aNSCs (blue bars). Categories having P

    Article Snippet: High throughput RNA sequencing (RNA-seq) To prepare samples for Illumina sequencing, 150–250 ng of RNA prepared from aNSCs or eNSCs (using the RNAeasy Plus Micro kit; Qiagen) was first treated to remove ribosomal RNA using the Ribo-Zero Magnetic Kit (Epicentre, Madison, WI), using the protocol for low input samples.

    Techniques: Expressing, Over Expression, RNA Sequencing Assay, Plasmid Preparation

    Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. ( A ) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. ( B ) Genes plotted in ( A ) were broken down according to their relative expression in eNSCs and aNSCs as indicated.

    Journal: Scientific Reports

    Article Title: Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells

    doi: 10.1038/s41598-018-25921-8

    Figure Lengend Snippet: Comparison of gene expression changes in embryonic and adult NSCs caused by Bmi1 overexpression. ( A ) Reads per kilobase per million reads (RPKM) from RNA-seq data from aNSCs and eSNCs is plotted for 4296 genes having expression altered at least two-fold by Bmi1 overexpression in either eNSCs or aNSCs. ( B ) Genes plotted in ( A ) were broken down according to their relative expression in eNSCs and aNSCs as indicated.

    Article Snippet: High throughput RNA sequencing (RNA-seq) To prepare samples for Illumina sequencing, 150–250 ng of RNA prepared from aNSCs or eNSCs (using the RNAeasy Plus Micro kit; Qiagen) was first treated to remove ribosomal RNA using the Ribo-Zero Magnetic Kit (Epicentre, Madison, WI), using the protocol for low input samples.

    Techniques: Expressing, Over Expression, RNA Sequencing Assay

    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