ribonucleoside vanadyl complex 200 mm  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    New England Biolabs ribonucleoside vanadyl complex 200 mm
    RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged <t>200</t> nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).
    Ribonucleoside Vanadyl Complex 200 Mm, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ribonucleoside vanadyl complex 200 mm/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ribonucleoside vanadyl complex 200 mm - by Bioz Stars, 2022-07
    97/100 stars

    Images

    1) Product Images from "Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes"

    Article Title: Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

    Journal: JCI Insight

    doi: 10.1172/jci.insight.122686

    RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged 200 nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).
    Figure Legend Snippet: RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged 200 nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).

    Techniques Used: Fluorescence In Situ Hybridization, Fluorescence, Labeling

    2) Product Images from "h-channels contribute to divergent electrophysiological properties of supragranular pyramidal neurons in human versus mouse cerebral cortex"

    Article Title: h-channels contribute to divergent electrophysiological properties of supragranular pyramidal neurons in human versus mouse cerebral cortex

    Journal: Neuron

    doi: 10.1016/j.neuron.2018.10.012

    HCN1–4 RNA expression in human versus mouse neocortex. A) Single nucleus HCN channel subunit mRNA expression in excitatory neurons from human temporal cortex arranged by layer. Violin plots represent distribution of mRNA expression on a log scale with a maximum counts per million (CPM) of introns + exons value of 4000. For reference, single nucleus HCN channel subunit mRNA expression in inhibitory neurons, aggregated across all layers, is also shown. B) Single cell HCN channel subunit mRNA expression in excitatory neurons from mouse visual cortex arranged by layer. The expression of SLC17A7 (magenta), GAD2 (turquoise), and HCN1 (yellow) was assessed by mFISH in C) mouse temporal association area (TeA), as well as D) human middle temporal gyrus (MTG). In layers 2 and 3 of mouse TeA (c1, c1’, c1’’, c2, c2’,c2’’,), Hcn1 expression in excitatory neurons is low compared with expression in L5 (c3, c3’,c3’’). HCN1 expression is high in Gad2 -expressing inhibitory cells in all layers of mouse TeA (note, L4 is not prominent in mouse TeA). In human MTG HCN1 expression is prominent in excitatory and inhibitory neurons in the supragranular and infragranular layers (d1-d3’’). Large white dots reflect fluorescence from lipofuscin. Images with ‘’ , are the same as images with ‘, but signals from each probe are color coded for clarity. E) RNA-counts per cell as a function of depth from pial surface in human and mouse. Scale bars in C,D = 200 μm. Scale bars in c1’-d3’’= 50 μm. Images in panels C and D are from composite images.
    Figure Legend Snippet: HCN1–4 RNA expression in human versus mouse neocortex. A) Single nucleus HCN channel subunit mRNA expression in excitatory neurons from human temporal cortex arranged by layer. Violin plots represent distribution of mRNA expression on a log scale with a maximum counts per million (CPM) of introns + exons value of 4000. For reference, single nucleus HCN channel subunit mRNA expression in inhibitory neurons, aggregated across all layers, is also shown. B) Single cell HCN channel subunit mRNA expression in excitatory neurons from mouse visual cortex arranged by layer. The expression of SLC17A7 (magenta), GAD2 (turquoise), and HCN1 (yellow) was assessed by mFISH in C) mouse temporal association area (TeA), as well as D) human middle temporal gyrus (MTG). In layers 2 and 3 of mouse TeA (c1, c1’, c1’’, c2, c2’,c2’’,), Hcn1 expression in excitatory neurons is low compared with expression in L5 (c3, c3’,c3’’). HCN1 expression is high in Gad2 -expressing inhibitory cells in all layers of mouse TeA (note, L4 is not prominent in mouse TeA). In human MTG HCN1 expression is prominent in excitatory and inhibitory neurons in the supragranular and infragranular layers (d1-d3’’). Large white dots reflect fluorescence from lipofuscin. Images with ‘’ , are the same as images with ‘, but signals from each probe are color coded for clarity. E) RNA-counts per cell as a function of depth from pial surface in human and mouse. Scale bars in C,D = 200 μm. Scale bars in c1’-d3’’= 50 μm. Images in panels C and D are from composite images.

    Techniques Used: RNA Expression, Expressing, Fluorescence

    3) Product Images from "Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection"

    Article Title: Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.853188

    Immuno-LCM-RNAseq of the human jejunum lymphatic vessel from a clinical, RNAlater-preserved jejunum tissue. (A) Left: Immunofluorescence image of the lymphatic vessels in the human jejunum tissue section using an anti-Podoplanin antibody (with 10 mM RVC). Right: bright field image after LCM. LC: lymphatic cells; Sm: submucosal layer; M: muscle layer (B) RNA quality of the leftover materials after LCM. (C) The quality of the cDNA library prepared from this RNA. Scale bar: 200 μm.
    Figure Legend Snippet: Immuno-LCM-RNAseq of the human jejunum lymphatic vessel from a clinical, RNAlater-preserved jejunum tissue. (A) Left: Immunofluorescence image of the lymphatic vessels in the human jejunum tissue section using an anti-Podoplanin antibody (with 10 mM RVC). Right: bright field image after LCM. LC: lymphatic cells; Sm: submucosal layer; M: muscle layer (B) RNA quality of the leftover materials after LCM. (C) The quality of the cDNA library prepared from this RNA. Scale bar: 200 μm.

    Techniques Used: Laser Capture Microdissection, Immunofluorescence, cDNA Library Assay

    High quality IF images obtained from various snap-frozen tissue sections with standard immunolabelling procedures in the presence of 10 mM RVC. The presence of RVC in the incubation solution apparently has no adverse effect on antibody-antigen interactions. Here, anti-Lyve1 labels lymphatic vessels and anti-PanCK labels epithelial cells. Scale bar: 200 μm (stomach, colon, testis), 100 μm (liver, kidney, spleen).
    Figure Legend Snippet: High quality IF images obtained from various snap-frozen tissue sections with standard immunolabelling procedures in the presence of 10 mM RVC. The presence of RVC in the incubation solution apparently has no adverse effect on antibody-antigen interactions. Here, anti-Lyve1 labels lymphatic vessels and anti-PanCK labels epithelial cells. Scale bar: 200 μm (stomach, colon, testis), 100 μm (liver, kidney, spleen).

    Techniques Used: Incubation

    4) Product Images from "Disruption in murine Eml1 perturbs retinal lamination during early development"

    Article Title: Disruption in murine Eml1 perturbs retinal lamination during early development

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62373-5

    The laminar organization of the retina is perturbed in homozygous Eml1 tvrm360 eyes. ( a ) Retinal sections of control eyes show dense hematoxylin-stained nuclei in the outer nuclear layer (ONL) at postnatal day (P) 8 and 1 M. In contrast, in Eml1 tvrm360 mutant retinas dense hematoxylin-stained nuclei were also observed in the GCL at P8 and in the INL at P8 and 1 M. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 µm. ( b–g ) Full field ERG responses recorded in Eml1 tvrm360 (n = 5) and control littermates (n = 5) at one month of age. (b) Mean scotopic traces from a representative wild-type (WT, black ) and homozygous Eml1 tvrm360 littermate ( tvrm360 , blue) with increasing light stimulus intensities (values indicate flash illuminance in log cd s m-2). Scale bars: vertical, 500 µV; horizontal, 50 ms. Light dose response analysis showing mean ± SEM scotopic ( c ) b-wave and ( d ) a-wave amplitudes of WT (black circles) and tvrm360 (blue circles) mice. ( e ) Representative photopic traces from the same mice as in ( b ). Scale bars: vertical, 200 µV; horizontal, 50 ms. ( f ) Light dose response analysis showing mean ± standard error photopic b-wave amplitudes in WT (black circles) and tvrm360 (blue circles) mice. Asterisks indicate significant values (Pairwise t-test; rod b-wave P
    Figure Legend Snippet: The laminar organization of the retina is perturbed in homozygous Eml1 tvrm360 eyes. ( a ) Retinal sections of control eyes show dense hematoxylin-stained nuclei in the outer nuclear layer (ONL) at postnatal day (P) 8 and 1 M. In contrast, in Eml1 tvrm360 mutant retinas dense hematoxylin-stained nuclei were also observed in the GCL at P8 and in the INL at P8 and 1 M. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 µm. ( b–g ) Full field ERG responses recorded in Eml1 tvrm360 (n = 5) and control littermates (n = 5) at one month of age. (b) Mean scotopic traces from a representative wild-type (WT, black ) and homozygous Eml1 tvrm360 littermate ( tvrm360 , blue) with increasing light stimulus intensities (values indicate flash illuminance in log cd s m-2). Scale bars: vertical, 500 µV; horizontal, 50 ms. Light dose response analysis showing mean ± SEM scotopic ( c ) b-wave and ( d ) a-wave amplitudes of WT (black circles) and tvrm360 (blue circles) mice. ( e ) Representative photopic traces from the same mice as in ( b ). Scale bars: vertical, 200 µV; horizontal, 50 ms. ( f ) Light dose response analysis showing mean ± standard error photopic b-wave amplitudes in WT (black circles) and tvrm360 (blue circles) mice. Asterisks indicate significant values (Pairwise t-test; rod b-wave P

    Techniques Used: Staining, Mutagenesis, Mouse Assay

    5) Product Images from "RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples"

    Article Title: RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0218-0

    RollFISH in FFPE breast cancer samples. a–c Expression of HER2, ER, and Ki-67 in breast cancer tissues. Pathological information for each of the samples shown is listed in Supplementary Tables 2 and 3 . The percentages above each column in b and c indicate the proportion of positive tumor cells as revealed by IHC. d The expression of HER2, ER, and Ki-67 across an entire FFPE tissue section of tumor #8 (see Supplementary Tables 2 and 3 ). The regions in dashed boxes (d3 and d4) in upper large images (first-row) are zoomed-in, and shown below as two rows of images with region names (d3 and d4). Scale bars, 200 µm (first-row images) and 30 µm (second- and third-row images). Blue, Nuclei. e Quantification of HER2, ER, and Ki-67 in regions d1 and d2 in d . Cells without any signals were excluded from the analysis. f Expression of HER2, ER, and Ki-67 in regions d3 and d4. In all the box plots presented in this figure, boxes extend from the 25th to the 75th percentile, whiskers extend from 2.5 to 97.5 percentiles, and the line inside each box represents the median value
    Figure Legend Snippet: RollFISH in FFPE breast cancer samples. a–c Expression of HER2, ER, and Ki-67 in breast cancer tissues. Pathological information for each of the samples shown is listed in Supplementary Tables 2 and 3 . The percentages above each column in b and c indicate the proportion of positive tumor cells as revealed by IHC. d The expression of HER2, ER, and Ki-67 across an entire FFPE tissue section of tumor #8 (see Supplementary Tables 2 and 3 ). The regions in dashed boxes (d3 and d4) in upper large images (first-row) are zoomed-in, and shown below as two rows of images with region names (d3 and d4). Scale bars, 200 µm (first-row images) and 30 µm (second- and third-row images). Blue, Nuclei. e Quantification of HER2, ER, and Ki-67 in regions d1 and d2 in d . Cells without any signals were excluded from the analysis. f Expression of HER2, ER, and Ki-67 in regions d3 and d4. In all the box plots presented in this figure, boxes extend from the 25th to the 75th percentile, whiskers extend from 2.5 to 97.5 percentiles, and the line inside each box represents the median value

    Techniques Used: Formalin-fixed Paraffin-Embedded, Expressing, Immunohistochemistry

    RollFISH in tissue microarrays. a Examples of HER2 levels in five different cores. Regions inside dashed boxes in first-row images are magnified below. Core names correspond to the position of the core in the TMA, as shown in Supplementary Fig. 9 . Blue, Nuclei. Scale bar, 200 µm (first-row images) or 25 µm (second row images). b HER2 levels by IHC group in one replicate experiment. Each dot corresponds to one sample. Error bars show the mean value ± s.d. for each IHC score group. Norm, normal breast tissues or adrenal gland. Neg, 0 for IHC HER2 score. c Correlation between RollFISH counts in two replicate experiments on two consecutive TMA sections. The black line represents the best linear regression fit. R 2 , Pearson’s correlation coefficient. Each dot represents a sample and samples were color-coded based on their HER2 IHC score. d Receiver operating characteristic curve of the sensitivity and specificity with which RollFISH correctly detects HER2-positive tumors previously classified based on IHC
    Figure Legend Snippet: RollFISH in tissue microarrays. a Examples of HER2 levels in five different cores. Regions inside dashed boxes in first-row images are magnified below. Core names correspond to the position of the core in the TMA, as shown in Supplementary Fig. 9 . Blue, Nuclei. Scale bar, 200 µm (first-row images) or 25 µm (second row images). b HER2 levels by IHC group in one replicate experiment. Each dot corresponds to one sample. Error bars show the mean value ± s.d. for each IHC score group. Norm, normal breast tissues or adrenal gland. Neg, 0 for IHC HER2 score. c Correlation between RollFISH counts in two replicate experiments on two consecutive TMA sections. The black line represents the best linear regression fit. R 2 , Pearson’s correlation coefficient. Each dot represents a sample and samples were color-coded based on their HER2 IHC score. d Receiver operating characteristic curve of the sensitivity and specificity with which RollFISH correctly detects HER2-positive tumors previously classified based on IHC

    Techniques Used: Immunohistochemistry

    6) Product Images from "Conserved cell types with divergent features in human versus mouse cortex"

    Article Title: Conserved cell types with divergent features in human versus mouse cortex

    Journal: Nature

    doi: 10.1038/s41586-019-1506-7

    Quantifying human and mouse cell type homology and comparing cell type frequencies between species. a-d , Heatmaps with inferred cell type homologies highlighted in blue boxes. For each pair of clusters, the shade of grey indicates the minimum proportion of samples that co-cluster. Homologies for human and mouse inhibitory neurons (a) , excitatory neurons (b) , and non-neuronal cells (c) were predicted based on shared cluster membership using mouse cells from two cortical areas (V1 and ALM) and two unsupervised alignment algorithms (scAlign and Seurat). d , Mouse V1 and mouse ALM excitatory neurons were aligned with scAlign. Blue boxes indicate V1 and ALM clusters that align to the same human clusters in b and are members of homologous cell types. Note that cell types can be matched at higher resolution within than between species, as expected. e , Left to right: violin plot (n=10,525 nuclei) showing expression of specific markers of the putative extratelencephalic (ET) EXC L4-5 FEZF2 SCN4B cluster (black box) and NPTX1 , a gene expressed by all non-PT excitatory neurons. Each row represents a gene, the black dots in each violin represent median gene expression within clusters, and the maximum expression value for each gene is shown on the right-hand side of each row. Expression values are shown on a linear scale. Representative inverted DAPI-stained cortical column (scale bar, 200 μm) with red dots marking the position of cells positive for the genes SLC17A7 and FAM84B and negative for NPTX1 illustrates the relative abundance of the EXC L4-5 FEZF2 SCN4B type in human MTG. Representative examples (arrows) of FAM84B (scale bar, 25 μm) and POU3F1 -expressing cells (scale bar, 25 μm). Expression of Fam84b in mouse TEa (scale bar, 75 μm) is shown in the adjacent panel. panel. f , mFISH for NPTX1 , a marker of non-PT excitatory types and SLC17A7 , shows that NPTX1 labels most SLC17A7+ cells across all cortical layers. Boxed region Boxed region shown at higher the magnification to the right. One SLC17A7+ cell (white arrow) cell (white arrow) is NPTX1− , but all other all other SLC17A7+ cells are NPTX1+ . Scale bars, left (200 μm), right (50 μm). Right: representative inverted DAPI-stained cortical column with red dots that represent SLC17A7+ , NPTX1− , and POU3F1+ cells. Scale bar, 200 μm. e, f, Experiments were repeated on 3 donors (human) and 2 mice with similar results. g , ISH validation of layer distributions in human MTG and mouse primary visual cortex (data from Tasic et al. 22 ). Cells are labeled by cluster marker genes in human ( RORB +/ CNR1 −/ PRSS12 +) and mouse ( Scnn1a +/ Hsd11b1+ ). ISH was performed on 3 human donors with similar results. For mouse, 1 experiment was performed.
    Figure Legend Snippet: Quantifying human and mouse cell type homology and comparing cell type frequencies between species. a-d , Heatmaps with inferred cell type homologies highlighted in blue boxes. For each pair of clusters, the shade of grey indicates the minimum proportion of samples that co-cluster. Homologies for human and mouse inhibitory neurons (a) , excitatory neurons (b) , and non-neuronal cells (c) were predicted based on shared cluster membership using mouse cells from two cortical areas (V1 and ALM) and two unsupervised alignment algorithms (scAlign and Seurat). d , Mouse V1 and mouse ALM excitatory neurons were aligned with scAlign. Blue boxes indicate V1 and ALM clusters that align to the same human clusters in b and are members of homologous cell types. Note that cell types can be matched at higher resolution within than between species, as expected. e , Left to right: violin plot (n=10,525 nuclei) showing expression of specific markers of the putative extratelencephalic (ET) EXC L4-5 FEZF2 SCN4B cluster (black box) and NPTX1 , a gene expressed by all non-PT excitatory neurons. Each row represents a gene, the black dots in each violin represent median gene expression within clusters, and the maximum expression value for each gene is shown on the right-hand side of each row. Expression values are shown on a linear scale. Representative inverted DAPI-stained cortical column (scale bar, 200 μm) with red dots marking the position of cells positive for the genes SLC17A7 and FAM84B and negative for NPTX1 illustrates the relative abundance of the EXC L4-5 FEZF2 SCN4B type in human MTG. Representative examples (arrows) of FAM84B (scale bar, 25 μm) and POU3F1 -expressing cells (scale bar, 25 μm). Expression of Fam84b in mouse TEa (scale bar, 75 μm) is shown in the adjacent panel. panel. f , mFISH for NPTX1 , a marker of non-PT excitatory types and SLC17A7 , shows that NPTX1 labels most SLC17A7+ cells across all cortical layers. Boxed region Boxed region shown at higher the magnification to the right. One SLC17A7+ cell (white arrow) cell (white arrow) is NPTX1− , but all other all other SLC17A7+ cells are NPTX1+ . Scale bars, left (200 μm), right (50 μm). Right: representative inverted DAPI-stained cortical column with red dots that represent SLC17A7+ , NPTX1− , and POU3F1+ cells. Scale bar, 200 μm. e, f, Experiments were repeated on 3 donors (human) and 2 mice with similar results. g , ISH validation of layer distributions in human MTG and mouse primary visual cortex (data from Tasic et al. 22 ). Cells are labeled by cluster marker genes in human ( RORB +/ CNR1 −/ PRSS12 +) and mouse ( Scnn1a +/ Hsd11b1+ ). ISH was performed on 3 human donors with similar results. For mouse, 1 experiment was performed.

    Techniques Used: Expressing, Staining, Marker, Mouse Assay, In Situ Hybridization, Labeling

    7) Product Images from "A novel lncRNA Discn fine-tunes replication protein A (RPA) availability to promote genomic stability"

    Article Title: A novel lncRNA Discn fine-tunes replication protein A (RPA) availability to promote genomic stability

    Journal: Nature Communications

    doi: 10.1038/s41467-021-25827-6

    RPA overexpression rescues genomic instability in Discn KD mESCs. RPA overexpression in Discn KD mESCs restored the ATR-CHK1 signaling ( a ), the stalled fork stability ( b ), and the fork restart ability ( c ). It also rescued the defects in dormant origin firing ( d ), ssDNA generation ( e ), and DSB formations ( f ) under HU treatment. In addition, RPA overexpression in Discn KD mESCs recovered the HR repair competence ( g ). Working model of Discn in safeguarding free RPA pool and genomic stability ( h ). Scale bar, 10 μm in ( e ) and 5 μm in ( g ). Three independent experiments were repeated with similar results in ( a ). At least 200 fibers from three independent replications were analyzed in ( b , c ). At least 50 continuous fibers from three independent replications were analyzed in ( d ). At least 19 images were analyzed in each condition in ( e ). Data were from three replications in ( f , g ). Data were representative of individual values with box and whiskers plots showing the median, upper and lower quartiles, and minimum and maximum in ( d , e ). Data were presented as mean ± SEM in ( c , f and g ). Statistical differences were determined using two-tailed Student’s t -test.
    Figure Legend Snippet: RPA overexpression rescues genomic instability in Discn KD mESCs. RPA overexpression in Discn KD mESCs restored the ATR-CHK1 signaling ( a ), the stalled fork stability ( b ), and the fork restart ability ( c ). It also rescued the defects in dormant origin firing ( d ), ssDNA generation ( e ), and DSB formations ( f ) under HU treatment. In addition, RPA overexpression in Discn KD mESCs recovered the HR repair competence ( g ). Working model of Discn in safeguarding free RPA pool and genomic stability ( h ). Scale bar, 10 μm in ( e ) and 5 μm in ( g ). Three independent experiments were repeated with similar results in ( a ). At least 200 fibers from three independent replications were analyzed in ( b , c ). At least 50 continuous fibers from three independent replications were analyzed in ( d ). At least 19 images were analyzed in each condition in ( e ). Data were from three replications in ( f , g ). Data were representative of individual values with box and whiskers plots showing the median, upper and lower quartiles, and minimum and maximum in ( d , e ). Data were presented as mean ± SEM in ( c , f and g ). Statistical differences were determined using two-tailed Student’s t -test.

    Techniques Used: Recombinase Polymerase Amplification, Over Expression, Two Tailed Test

    Discn regulates DNA replication stress response and HR-mediated DNA DSB repair. a Mouse ESCs were treated with hydroxyurea (HU) to induce replication stress. ATR-CHK1 kinase activation failed to be well sustained in two Discn knockdown cells (KD-1 and KD-2) when compared to the knockdown control (KD-C). Three independent experiments were repeated with similar results. b DNA fiber assay revealed that the stalled forks in Discn KD cells were prone to undergo degradation after HU treatment. 200 fibers from three independent replications were analyzed. c Discn KD compromised the stalled fork restart. At least 200 fibers from three independent replications were analyzed. d Discn KD enhanced dormant origin firing as indicated by the reduced mean fork spacing. At least 50 continuous fibers from three independent replications were analyzed. e Discn KD increased the content of ssDNA as measured by the native BrdU incorporation assay at S phase. Scale bar, 10 μm. Quantification is shown on the right. At least 20 images were analyzed in each condition. f Compared to control ESCs (KD-C), Discn KD cells were more sensitive to hydroxyurea treatment and accumulated more DNA DSBs as measured by neutral comet assay. Data were from three replications and shown as mean ± SEM. g FACS analysis of reporter ESCs showed that Discn KD suppressed HR-mediated DNA DSB repair, but inversely stimulated NHEJ repair pathway. Data were from three independent experiments. h DNA DSBs were generated by laser microirradiation. Discn KD impaired the recruitment of Rad51 to DSB sites labeled with γH2AX at S phase (BrdU + ), indicating the suppression of HR pathway. Right panel showed the proportions of S phase cells capable of HR repair (three replicates, 50 cells in each replicate). Scale bar, 5 μm. i Immunoblotting showed a decrease in chromatin-bound Rad51 in Discn KD ESCs synchronized at S phase. Three independent experiments were repeated with similar results. b , c , f – h Data were shown as mean ± SEM, two-tailed Student’s t -test. d , e Data were representative of individual values with box and whiskers plots showing the median, upper and lower quartiles, and minimum and maximum. Two-tailed Student’s t -test.
    Figure Legend Snippet: Discn regulates DNA replication stress response and HR-mediated DNA DSB repair. a Mouse ESCs were treated with hydroxyurea (HU) to induce replication stress. ATR-CHK1 kinase activation failed to be well sustained in two Discn knockdown cells (KD-1 and KD-2) when compared to the knockdown control (KD-C). Three independent experiments were repeated with similar results. b DNA fiber assay revealed that the stalled forks in Discn KD cells were prone to undergo degradation after HU treatment. 200 fibers from three independent replications were analyzed. c Discn KD compromised the stalled fork restart. At least 200 fibers from three independent replications were analyzed. d Discn KD enhanced dormant origin firing as indicated by the reduced mean fork spacing. At least 50 continuous fibers from three independent replications were analyzed. e Discn KD increased the content of ssDNA as measured by the native BrdU incorporation assay at S phase. Scale bar, 10 μm. Quantification is shown on the right. At least 20 images were analyzed in each condition. f Compared to control ESCs (KD-C), Discn KD cells were more sensitive to hydroxyurea treatment and accumulated more DNA DSBs as measured by neutral comet assay. Data were from three replications and shown as mean ± SEM. g FACS analysis of reporter ESCs showed that Discn KD suppressed HR-mediated DNA DSB repair, but inversely stimulated NHEJ repair pathway. Data were from three independent experiments. h DNA DSBs were generated by laser microirradiation. Discn KD impaired the recruitment of Rad51 to DSB sites labeled with γH2AX at S phase (BrdU + ), indicating the suppression of HR pathway. Right panel showed the proportions of S phase cells capable of HR repair (three replicates, 50 cells in each replicate). Scale bar, 5 μm. i Immunoblotting showed a decrease in chromatin-bound Rad51 in Discn KD ESCs synchronized at S phase. Three independent experiments were repeated with similar results. b , c , f – h Data were shown as mean ± SEM, two-tailed Student’s t -test. d , e Data were representative of individual values with box and whiskers plots showing the median, upper and lower quartiles, and minimum and maximum. Two-tailed Student’s t -test.

    Techniques Used: Activation Assay, BrdU Incorporation Assay, Neutral Comet Assay, FACS, Non-Homologous End Joining, Generated, Labeling, Two Tailed Test

    8) Product Images from "Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level"

    Article Title: Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22070-x

    MAMP sensing in epithelial monolayers shows digital and analog components. a Schematic detailing the NF-κB-dependent gene expression reporter system. An innate immune signal activates IKK which phosphorylates IκBα and sends it for degradation, freeing the fluorescently tagged NF-κB subunit (p65-mRuby) to enter the nucleus and transcribe the Venus-PEST reporter. b MCF10A NF-κB reporter cells were incubated in the presence or absence of IL-1β (10 ng/ml) and/or IKK inhibitor VII (10 µM). Representative images from five replicates taken at indicated times are shown. Scale bar, 50 μm. c Representative single-cell trace. The median intensity of the nuclear/cytoplasmic NF-κB is plotted in black, while the median nuclear + cytoplasmic intensity gene expression reporter is in green. d Schematic of innate immune receptors and inputs that signal to NF-κB. TLR5, IL1R, and TLR1/2 all signal through MyD88. e Heatmap of all cells in the MAMP screen. MCF10A monoclonal reporter cell line was imaged for 50 min then treated with the indicated input and concentration. Concentrations at 100x were 1 µg/ml (Flagellin), 200 µg/ml (Poly(I:C)), 1 µg/ml (Pam3CSK4), 1 µg/ml (TNFα), and 100 ng/ml (IL-1β). Heatmaps are ordered top to bottom from highest to lowest maximum Venus expression. f (Top) Swarmplots of the area under the curve after the first NF-κB translocation peak (AUC steady) in 200 cells randomly sampled from the indicated input and concentration. (Bottom) Swarmplots of the 95th percentile of the gene expression in 200 cells randomly sampled from the indicated input and concentration. R squared values of the linear regression model fit to responses across the log of three concentrations. g Representative images of clonal MCF10A after 8 h of incubation with indicated stimulus (1 µg/ml (Flagellin), 100 ng/ml (IL-1β), 20 µg/ml (Poly(I:C)), 100 ng/ml (TNFα), and 1 µg/ml (Pam3CSK4)). H2B-iRFP nuclear marker is shown in red and Venus gene expression reporter is shown in cyan. Scale bar, 200 µm. h MCF10A NF-κB reporter cells stimulated with 1 µg/ml Pam3CSK4. Traces of NF-κB translocation (gray) and Venus expression (green) for five non-responders and responders are shown.
    Figure Legend Snippet: MAMP sensing in epithelial monolayers shows digital and analog components. a Schematic detailing the NF-κB-dependent gene expression reporter system. An innate immune signal activates IKK which phosphorylates IκBα and sends it for degradation, freeing the fluorescently tagged NF-κB subunit (p65-mRuby) to enter the nucleus and transcribe the Venus-PEST reporter. b MCF10A NF-κB reporter cells were incubated in the presence or absence of IL-1β (10 ng/ml) and/or IKK inhibitor VII (10 µM). Representative images from five replicates taken at indicated times are shown. Scale bar, 50 μm. c Representative single-cell trace. The median intensity of the nuclear/cytoplasmic NF-κB is plotted in black, while the median nuclear + cytoplasmic intensity gene expression reporter is in green. d Schematic of innate immune receptors and inputs that signal to NF-κB. TLR5, IL1R, and TLR1/2 all signal through MyD88. e Heatmap of all cells in the MAMP screen. MCF10A monoclonal reporter cell line was imaged for 50 min then treated with the indicated input and concentration. Concentrations at 100x were 1 µg/ml (Flagellin), 200 µg/ml (Poly(I:C)), 1 µg/ml (Pam3CSK4), 1 µg/ml (TNFα), and 100 ng/ml (IL-1β). Heatmaps are ordered top to bottom from highest to lowest maximum Venus expression. f (Top) Swarmplots of the area under the curve after the first NF-κB translocation peak (AUC steady) in 200 cells randomly sampled from the indicated input and concentration. (Bottom) Swarmplots of the 95th percentile of the gene expression in 200 cells randomly sampled from the indicated input and concentration. R squared values of the linear regression model fit to responses across the log of three concentrations. g Representative images of clonal MCF10A after 8 h of incubation with indicated stimulus (1 µg/ml (Flagellin), 100 ng/ml (IL-1β), 20 µg/ml (Poly(I:C)), 100 ng/ml (TNFα), and 1 µg/ml (Pam3CSK4)). H2B-iRFP nuclear marker is shown in red and Venus gene expression reporter is shown in cyan. Scale bar, 200 µm. h MCF10A NF-κB reporter cells stimulated with 1 µg/ml Pam3CSK4. Traces of NF-κB translocation (gray) and Venus expression (green) for five non-responders and responders are shown.

    Techniques Used: Expressing, Incubation, Concentration Assay, Translocation Assay, Marker

    9) Product Images from "Single-cell analysis of transcription kinetics across the cell cycle"

    Article Title: Single-cell analysis of transcription kinetics across the cell cycle

    Journal: eLife

    doi: 10.7554/eLife.12175

    Agreement between methods of measuring dosage compensation. The extracted fold change in nascent Oct4 (left) and Nanog (right) mRNA following gene replication, as measured using two methods: Method #1, comparing the mean number of nascent mRNA of cells in G1 phase to that of cells in G2 phase (see Figure 1F ). Error bars represent SE.M. from 3 experiments with > 200 cells per phase in each experiment. Method #2, extracting the fold change from a step-function fit to the nascent mRNA over time (see Figure 3B ). Error bars represent SEM from 3 experiments with > 600 cells per experiment. DOI: http://dx.doi.org/10.7554/eLife.12175.014
    Figure Legend Snippet: Agreement between methods of measuring dosage compensation. The extracted fold change in nascent Oct4 (left) and Nanog (right) mRNA following gene replication, as measured using two methods: Method #1, comparing the mean number of nascent mRNA of cells in G1 phase to that of cells in G2 phase (see Figure 1F ). Error bars represent SE.M. from 3 experiments with > 200 cells per phase in each experiment. Method #2, extracting the fold change from a step-function fit to the nascent mRNA over time (see Figure 3B ). Error bars represent SEM from 3 experiments with > 600 cells per experiment. DOI: http://dx.doi.org/10.7554/eLife.12175.014

    Techniques Used:

    Nascent mRNA correlation between two gene copies. ( A ) Heat maps of the number of nascent mRNA at the two gene copies within the same cell. Left, Oct4 (1 experiment with > 200 cells). Right, Nanog (1 experiment with > 200 cells). The Pearson’s correlation coefficient ( r ; mean ± SEM from 3 experiments with > 200 cells per experiment) between gene copies is indicated on each plot, as well as the p-value (mean ± SEM from 3 experiments with > 200 cells per experiment) obtained using a Student’s t-distribution (calculated using the MATLAB function corr). ( B ) The data in Panel A were reshuffled by pairing the nascent mRNA at one gene copy from a given cell with the nascent mRNA from a gene copy at another, randomly selected cell. DOI: http://dx.doi.org/10.7554/eLife.12175.010
    Figure Legend Snippet: Nascent mRNA correlation between two gene copies. ( A ) Heat maps of the number of nascent mRNA at the two gene copies within the same cell. Left, Oct4 (1 experiment with > 200 cells). Right, Nanog (1 experiment with > 200 cells). The Pearson’s correlation coefficient ( r ; mean ± SEM from 3 experiments with > 200 cells per experiment) between gene copies is indicated on each plot, as well as the p-value (mean ± SEM from 3 experiments with > 200 cells per experiment) obtained using a Student’s t-distribution (calculated using the MATLAB function corr). ( B ) The data in Panel A were reshuffled by pairing the nascent mRNA at one gene copy from a given cell with the nascent mRNA from a gene copy at another, randomly selected cell. DOI: http://dx.doi.org/10.7554/eLife.12175.010

    Techniques Used:

    10) Product Images from "Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level"

    Article Title: Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22070-x

    MAMP sensing in epithelial monolayers shows digital and analog components. a Schematic detailing the NF-κB-dependent gene expression reporter system. An innate immune signal activates IKK which phosphorylates IκBα and sends it for degradation, freeing the fluorescently tagged NF-κB subunit (p65-mRuby) to enter the nucleus and transcribe the Venus-PEST reporter. b MCF10A NF-κB reporter cells were incubated in the presence or absence of IL-1β (10 ng/ml) and/or IKK inhibitor VII (10 µM). Representative images from five replicates taken at indicated times are shown. Scale bar, 50 μm. c Representative single-cell trace. The median intensity of the nuclear/cytoplasmic NF-κB is plotted in black, while the median nuclear + cytoplasmic intensity gene expression reporter is in green. d Schematic of innate immune receptors and inputs that signal to NF-κB. TLR5, IL1R, and TLR1/2 all signal through MyD88. e Heatmap of all cells in the MAMP screen. MCF10A monoclonal reporter cell line was imaged for 50 min then treated with the indicated input and concentration. Concentrations at 100x were 1 µg/ml (Flagellin), 200 µg/ml (Poly(I:C)), 1 µg/ml (Pam3CSK4), 1 µg/ml (TNFα), and 100 ng/ml (IL-1β). Heatmaps are ordered top to bottom from highest to lowest maximum Venus expression. f (Top) Swarmplots of the area under the curve after the first NF-κB translocation peak (AUC steady) in 200 cells randomly sampled from the indicated input and concentration. (Bottom) Swarmplots of the 95th percentile of the gene expression in 200 cells randomly sampled from the indicated input and concentration. R squared values of the linear regression model fit to responses across the log of three concentrations. g Representative images of clonal MCF10A after 8 h of incubation with indicated stimulus (1 µg/ml (Flagellin), 100 ng/ml (IL-1β), 20 µg/ml (Poly(I:C)), 100 ng/ml (TNFα), and 1 µg/ml (Pam3CSK4)). H2B-iRFP nuclear marker is shown in red and Venus gene expression reporter is shown in cyan. Scale bar, 200 µm. h MCF10A NF-κB reporter cells stimulated with 1 µg/ml Pam3CSK4. Traces of NF-κB translocation (gray) and Venus expression (green) for five non-responders and responders are shown.
    Figure Legend Snippet: MAMP sensing in epithelial monolayers shows digital and analog components. a Schematic detailing the NF-κB-dependent gene expression reporter system. An innate immune signal activates IKK which phosphorylates IκBα and sends it for degradation, freeing the fluorescently tagged NF-κB subunit (p65-mRuby) to enter the nucleus and transcribe the Venus-PEST reporter. b MCF10A NF-κB reporter cells were incubated in the presence or absence of IL-1β (10 ng/ml) and/or IKK inhibitor VII (10 µM). Representative images from five replicates taken at indicated times are shown. Scale bar, 50 μm. c Representative single-cell trace. The median intensity of the nuclear/cytoplasmic NF-κB is plotted in black, while the median nuclear + cytoplasmic intensity gene expression reporter is in green. d Schematic of innate immune receptors and inputs that signal to NF-κB. TLR5, IL1R, and TLR1/2 all signal through MyD88. e Heatmap of all cells in the MAMP screen. MCF10A monoclonal reporter cell line was imaged for 50 min then treated with the indicated input and concentration. Concentrations at 100x were 1 µg/ml (Flagellin), 200 µg/ml (Poly(I:C)), 1 µg/ml (Pam3CSK4), 1 µg/ml (TNFα), and 100 ng/ml (IL-1β). Heatmaps are ordered top to bottom from highest to lowest maximum Venus expression. f (Top) Swarmplots of the area under the curve after the first NF-κB translocation peak (AUC steady) in 200 cells randomly sampled from the indicated input and concentration. (Bottom) Swarmplots of the 95th percentile of the gene expression in 200 cells randomly sampled from the indicated input and concentration. R squared values of the linear regression model fit to responses across the log of three concentrations. g Representative images of clonal MCF10A after 8 h of incubation with indicated stimulus (1 µg/ml (Flagellin), 100 ng/ml (IL-1β), 20 µg/ml (Poly(I:C)), 100 ng/ml (TNFα), and 1 µg/ml (Pam3CSK4)). H2B-iRFP nuclear marker is shown in red and Venus gene expression reporter is shown in cyan. Scale bar, 200 µm. h MCF10A NF-κB reporter cells stimulated with 1 µg/ml Pam3CSK4. Traces of NF-κB translocation (gray) and Venus expression (green) for five non-responders and responders are shown.

    Techniques Used: Expressing, Incubation, Concentration Assay, Translocation Assay, Marker

    11) Product Images from "Early alphavirus replication dynamics in single cells reveal a passive basis for superinfection exclusion"

    Article Title: Early alphavirus replication dynamics in single cells reveal a passive basis for superinfection exclusion

    Journal: bioRxiv

    doi: 10.1101/2020.09.07.282053

    Single cell measurements of superinfection exclusion reveal bidirectional viral competition. A) Cells initially infected with MOI 10 of SINV/nsP3-mTurquoise2 are subsequently infected with SINV/nsP3-mCitrine at a range of MOIs (0, 5, 10, or 30) after different delays (either 0m, 20m, 40m, or 60m). Representative images show a nuclear stain (red), SINV/nsP3-mCitrine (yellow) and SINV/nsP3-mTurquoise2 (blue) under each condition described. Scale bar is 100um. B) Total fluorescence per area (or MFI) averaged over all cells in the given condition as a function of delay (ΔT), shown as mean±SEM. Color coding of each line represents a different superinfecting MOI. C) A plot of the fraction of cells infected by the second virus, SINV/nsP3-mCitrine as a function of delay (ΔT) and superinfecting MOI. Error bars are bootstrapped SEM. D) Information from B and C are combined to visually depict how temporal delays and relative MOIs together influence superinfection. Color represents the average normalized fluorescence intensity, while circle size represents the fraction of cells infected. E-F) Single cell traces of replication kinetics from mTurquoise2 (left) and mCitrine (right) reporter viruses under the experimental conditions described in A through the 13h time course. Y-axes of subplots in E are all equal, as are the Y-axes in F MOIs indicated are of the superinfecting SINV/nsP3-mCitrine. The first virus is always kept at MOI 10. G) Total fluorescence averaged over single-cells of nsP3-mTurquoise2 versus nsP3-mCitrine at the end of the superinfection movie (13hpi) shown in E and F Circle size represents delay between the first and second viruses, and color represents the MOI of the second virus, each shown at mean±SEM. Gray line is a linear-least squares fit, with a Pearson correlation coefficient of r=-0.99. H) Lotka-Volterra simulations of the superinfection experiment where both the first and second viruses are modeled identically (r=0.03/min and K=200,000, see Methods), over a range of delays and superinfecting MOIs. Inset shows a model of viral kinetics, where both begin at MOI 10, but with ΔT=20min.
    Figure Legend Snippet: Single cell measurements of superinfection exclusion reveal bidirectional viral competition. A) Cells initially infected with MOI 10 of SINV/nsP3-mTurquoise2 are subsequently infected with SINV/nsP3-mCitrine at a range of MOIs (0, 5, 10, or 30) after different delays (either 0m, 20m, 40m, or 60m). Representative images show a nuclear stain (red), SINV/nsP3-mCitrine (yellow) and SINV/nsP3-mTurquoise2 (blue) under each condition described. Scale bar is 100um. B) Total fluorescence per area (or MFI) averaged over all cells in the given condition as a function of delay (ΔT), shown as mean±SEM. Color coding of each line represents a different superinfecting MOI. C) A plot of the fraction of cells infected by the second virus, SINV/nsP3-mCitrine as a function of delay (ΔT) and superinfecting MOI. Error bars are bootstrapped SEM. D) Information from B and C are combined to visually depict how temporal delays and relative MOIs together influence superinfection. Color represents the average normalized fluorescence intensity, while circle size represents the fraction of cells infected. E-F) Single cell traces of replication kinetics from mTurquoise2 (left) and mCitrine (right) reporter viruses under the experimental conditions described in A through the 13h time course. Y-axes of subplots in E are all equal, as are the Y-axes in F MOIs indicated are of the superinfecting SINV/nsP3-mCitrine. The first virus is always kept at MOI 10. G) Total fluorescence averaged over single-cells of nsP3-mTurquoise2 versus nsP3-mCitrine at the end of the superinfection movie (13hpi) shown in E and F Circle size represents delay between the first and second viruses, and color represents the MOI of the second virus, each shown at mean±SEM. Gray line is a linear-least squares fit, with a Pearson correlation coefficient of r=-0.99. H) Lotka-Volterra simulations of the superinfection experiment where both the first and second viruses are modeled identically (r=0.03/min and K=200,000, see Methods), over a range of delays and superinfecting MOIs. Inset shows a model of viral kinetics, where both begin at MOI 10, but with ΔT=20min.

    Techniques Used: Infection, Staining, Fluorescence

    12) Product Images from "A temporally controlled sequence of X-chromosome inactivation and reactivation defines female mouse in vitro germ cells with meiotic potential"

    Article Title: A temporally controlled sequence of X-chromosome inactivation and reactivation defines female mouse in vitro germ cells with meiotic potential

    Journal: bioRxiv

    doi: 10.1101/2021.08.11.455976

    Gene expression analysis reveals two PGCLCs subpopulations. (A) Top panel shows representative contour plots of FACS analysis of PGCLC induction, without or with Dox, in PGCLC d5. The number indicates the gated germ cells identified by CD61 and SSEA1 signal. Bottom panel indicates the percentages of X-active (green box) or X-inactive (red box) cells. For the +Dox condition, percentages of X-active and X-inactive originating from SSEA1+/CD61+ cells are shown. Shown are contour plots gated on live cells. (B) PCA of gene expression dynamics during PGCLC differentiation. n = top 500 most variable genes excluding X chromosomal genes. Axes indicate the variance. Arrows indicate hypothetical trajectory. Shapes indicate the clones (A11 = square, E9 = rhombus). (C) Representative images showing the X-activity reporter status in colonies formed by ESCs, XGFP+ PGCLCs and XGFP-PGCLCs after 7 days of culture in 2i/LIF on immortalized feeder cells. BF = bright field. Scale bar 200 μm. (D) FACS analysis showing the X-reporter status of the indicated cell types after 7 days of culture in 2i/LIF on immortalized feeder cells. Numbers indicate the percentage of cells falling in the corresponding gate. Histograms come from XTomato+ gated cells depleted of immortalized feeder cells.
    Figure Legend Snippet: Gene expression analysis reveals two PGCLCs subpopulations. (A) Top panel shows representative contour plots of FACS analysis of PGCLC induction, without or with Dox, in PGCLC d5. The number indicates the gated germ cells identified by CD61 and SSEA1 signal. Bottom panel indicates the percentages of X-active (green box) or X-inactive (red box) cells. For the +Dox condition, percentages of X-active and X-inactive originating from SSEA1+/CD61+ cells are shown. Shown are contour plots gated on live cells. (B) PCA of gene expression dynamics during PGCLC differentiation. n = top 500 most variable genes excluding X chromosomal genes. Axes indicate the variance. Arrows indicate hypothetical trajectory. Shapes indicate the clones (A11 = square, E9 = rhombus). (C) Representative images showing the X-activity reporter status in colonies formed by ESCs, XGFP+ PGCLCs and XGFP-PGCLCs after 7 days of culture in 2i/LIF on immortalized feeder cells. BF = bright field. Scale bar 200 μm. (D) FACS analysis showing the X-reporter status of the indicated cell types after 7 days of culture in 2i/LIF on immortalized feeder cells. Numbers indicate the percentage of cells falling in the corresponding gate. Histograms come from XTomato+ gated cells depleted of immortalized feeder cells.

    Techniques Used: Expressing, FACS, Clone Assay, Activity Assay

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    New England Biolabs ribonucleoside vanadyl complex 200 mm
    RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged <t>200</t> nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).
    Ribonucleoside Vanadyl Complex 200 Mm, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ribonucleoside vanadyl complex 200 mm/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ribonucleoside vanadyl complex 200 mm - by Bioz Stars, 2022-07
    97/100 stars
      Buy from Supplier

    Image Search Results


    RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged 200 nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).

    Journal: JCI Insight

    Article Title: Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

    doi: 10.1172/jci.insight.122686

    Figure Lengend Snippet: RNA foci tightly colocalized with MBNL1 clusters in DM1 cardiomyocytes. ( A and B ) Using probes specific to the RNA repeat expansions, FISH was used to monitor RNA foci and their proximity to MBNL using total internal reflection fluorescence. The cells were colabeled with an antibody to MBNL1 (green). Control iPSC-CMs were labeled with a repeat probe for DM1 (CAG) 5 in A and for DM2 (CAGG) 10 in B . The distance between RNA foci and MBNL1 foci was quantified. Because MBNL1 foci were only readily detected in DM1 cardiomyocytes, the data from control and DM2 cells represent background signal. Thus, this method was used to measure the distance between RNA repeat expansions and MBNL1 in DM1. ( A ) RNA foci colocalized with MBNL1 foci in DM1 cardiomyocytes, and the distance between RNA foci and MBNL1 averaged 200 nm, consistent with a very close physical association between RNA repeat expansions and MBNL. Control cardiomyocytes displayed a random distribution of distances (top panel), reflecting the absence of RNA foci and MBNL foci. ( B ) In DM2 cardiomyocytes, there was no colocalization of RNA foci with MBNL foci, reflecting the absence of MBNL clusters and a pattern similar to cells from healthy controls. Correspondingly, the distances were randomly distributed, similar to control iPSC-CMs (similar distribution between top and bottom panels). Scale bar: 5 μm (left);1 μm (right).

    Article Snippet: Hybridization was done in 2× SSC buffer with 30% formamide (MilliporeSigma, F7503), 0.02% BSA (MilliporeSigma, A7906), 66 μg/ml yeast tRNA (Thermo Fisher Scientific, 15401-011), 10% dextran sulfate (MilliporeSigma, D8906), and 2 mM vanadyl ribonucleoside complex (New England Biolabs, S1402S).

    Techniques: Fluorescence In Situ Hybridization, Fluorescence, Labeling

    HCN1–4 RNA expression in human versus mouse neocortex. A) Single nucleus HCN channel subunit mRNA expression in excitatory neurons from human temporal cortex arranged by layer. Violin plots represent distribution of mRNA expression on a log scale with a maximum counts per million (CPM) of introns + exons value of 4000. For reference, single nucleus HCN channel subunit mRNA expression in inhibitory neurons, aggregated across all layers, is also shown. B) Single cell HCN channel subunit mRNA expression in excitatory neurons from mouse visual cortex arranged by layer. The expression of SLC17A7 (magenta), GAD2 (turquoise), and HCN1 (yellow) was assessed by mFISH in C) mouse temporal association area (TeA), as well as D) human middle temporal gyrus (MTG). In layers 2 and 3 of mouse TeA (c1, c1’, c1’’, c2, c2’,c2’’,), Hcn1 expression in excitatory neurons is low compared with expression in L5 (c3, c3’,c3’’). HCN1 expression is high in Gad2 -expressing inhibitory cells in all layers of mouse TeA (note, L4 is not prominent in mouse TeA). In human MTG HCN1 expression is prominent in excitatory and inhibitory neurons in the supragranular and infragranular layers (d1-d3’’). Large white dots reflect fluorescence from lipofuscin. Images with ‘’ , are the same as images with ‘, but signals from each probe are color coded for clarity. E) RNA-counts per cell as a function of depth from pial surface in human and mouse. Scale bars in C,D = 200 μm. Scale bars in c1’-d3’’= 50 μm. Images in panels C and D are from composite images.

    Journal: Neuron

    Article Title: h-channels contribute to divergent electrophysiological properties of supragranular pyramidal neurons in human versus mouse cerebral cortex

    doi: 10.1016/j.neuron.2018.10.012

    Figure Lengend Snippet: HCN1–4 RNA expression in human versus mouse neocortex. A) Single nucleus HCN channel subunit mRNA expression in excitatory neurons from human temporal cortex arranged by layer. Violin plots represent distribution of mRNA expression on a log scale with a maximum counts per million (CPM) of introns + exons value of 4000. For reference, single nucleus HCN channel subunit mRNA expression in inhibitory neurons, aggregated across all layers, is also shown. B) Single cell HCN channel subunit mRNA expression in excitatory neurons from mouse visual cortex arranged by layer. The expression of SLC17A7 (magenta), GAD2 (turquoise), and HCN1 (yellow) was assessed by mFISH in C) mouse temporal association area (TeA), as well as D) human middle temporal gyrus (MTG). In layers 2 and 3 of mouse TeA (c1, c1’, c1’’, c2, c2’,c2’’,), Hcn1 expression in excitatory neurons is low compared with expression in L5 (c3, c3’,c3’’). HCN1 expression is high in Gad2 -expressing inhibitory cells in all layers of mouse TeA (note, L4 is not prominent in mouse TeA). In human MTG HCN1 expression is prominent in excitatory and inhibitory neurons in the supragranular and infragranular layers (d1-d3’’). Large white dots reflect fluorescence from lipofuscin. Images with ‘’ , are the same as images with ‘, but signals from each probe are color coded for clarity. E) RNA-counts per cell as a function of depth from pial surface in human and mouse. Scale bars in C,D = 200 μm. Scale bars in c1’-d3’’= 50 μm. Images in panels C and D are from composite images.

    Article Snippet: Sections were incubated in hybridization buffer (10% Formamide (v/v, Sigma Aldrich 4650), 10% Dextran Sulfate (w/v, Sigma Aldrich D8906), 200 μg/mL BSA (Ambion AM2616), 2 mM Ribonucleoside vanadyl complex (New England Biolabs, S1402S), 1mg/mL tRNA (Sigma 10109541001) in 2XSSC) for 5 min at 38.5 C. Probes were designed antisense to the following genes: HCN1 , HCN2 , HCN3 , HCN4 , SLC17A7 , GAD2 and PEXL5 ( ).

    Techniques: RNA Expression, Expressing, Fluorescence

    Immuno-LCM-RNAseq of the human jejunum lymphatic vessel from a clinical, RNAlater-preserved jejunum tissue. (A) Left: Immunofluorescence image of the lymphatic vessels in the human jejunum tissue section using an anti-Podoplanin antibody (with 10 mM RVC). Right: bright field image after LCM. LC: lymphatic cells; Sm: submucosal layer; M: muscle layer (B) RNA quality of the leftover materials after LCM. (C) The quality of the cDNA library prepared from this RNA. Scale bar: 200 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection

    doi: 10.3389/fcell.2022.853188

    Figure Lengend Snippet: Immuno-LCM-RNAseq of the human jejunum lymphatic vessel from a clinical, RNAlater-preserved jejunum tissue. (A) Left: Immunofluorescence image of the lymphatic vessels in the human jejunum tissue section using an anti-Podoplanin antibody (with 10 mM RVC). Right: bright field image after LCM. LC: lymphatic cells; Sm: submucosal layer; M: muscle layer (B) RNA quality of the leftover materials after LCM. (C) The quality of the cDNA library prepared from this RNA. Scale bar: 200 μm.

    Article Snippet: The fixed sections were then dried in the cryostat for 5 min, and washed 3 times with ice-cold 10 mM Ribonucleoside Vanadyl Complex (RVC) (New England BioLabs, cat. no. S1402S, Ipswich, Mass, United States) in buffer A (10 mM NaCl, 3 mM MgCl2, 20 mM Tris•HCl, pH 7.4) in a RNA-specific biological safety cabinet.

    Techniques: Laser Capture Microdissection, Immunofluorescence, cDNA Library Assay

    High quality IF images obtained from various snap-frozen tissue sections with standard immunolabelling procedures in the presence of 10 mM RVC. The presence of RVC in the incubation solution apparently has no adverse effect on antibody-antigen interactions. Here, anti-Lyve1 labels lymphatic vessels and anti-PanCK labels epithelial cells. Scale bar: 200 μm (stomach, colon, testis), 100 μm (liver, kidney, spleen).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection

    doi: 10.3389/fcell.2022.853188

    Figure Lengend Snippet: High quality IF images obtained from various snap-frozen tissue sections with standard immunolabelling procedures in the presence of 10 mM RVC. The presence of RVC in the incubation solution apparently has no adverse effect on antibody-antigen interactions. Here, anti-Lyve1 labels lymphatic vessels and anti-PanCK labels epithelial cells. Scale bar: 200 μm (stomach, colon, testis), 100 μm (liver, kidney, spleen).

    Article Snippet: The fixed sections were then dried in the cryostat for 5 min, and washed 3 times with ice-cold 10 mM Ribonucleoside Vanadyl Complex (RVC) (New England BioLabs, cat. no. S1402S, Ipswich, Mass, United States) in buffer A (10 mM NaCl, 3 mM MgCl2, 20 mM Tris•HCl, pH 7.4) in a RNA-specific biological safety cabinet.

    Techniques: Incubation

    The laminar organization of the retina is perturbed in homozygous Eml1 tvrm360 eyes. ( a ) Retinal sections of control eyes show dense hematoxylin-stained nuclei in the outer nuclear layer (ONL) at postnatal day (P) 8 and 1 M. In contrast, in Eml1 tvrm360 mutant retinas dense hematoxylin-stained nuclei were also observed in the GCL at P8 and in the INL at P8 and 1 M. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 µm. ( b–g ) Full field ERG responses recorded in Eml1 tvrm360 (n = 5) and control littermates (n = 5) at one month of age. (b) Mean scotopic traces from a representative wild-type (WT, black ) and homozygous Eml1 tvrm360 littermate ( tvrm360 , blue) with increasing light stimulus intensities (values indicate flash illuminance in log cd s m-2). Scale bars: vertical, 500 µV; horizontal, 50 ms. Light dose response analysis showing mean ± SEM scotopic ( c ) b-wave and ( d ) a-wave amplitudes of WT (black circles) and tvrm360 (blue circles) mice. ( e ) Representative photopic traces from the same mice as in ( b ). Scale bars: vertical, 200 µV; horizontal, 50 ms. ( f ) Light dose response analysis showing mean ± standard error photopic b-wave amplitudes in WT (black circles) and tvrm360 (blue circles) mice. Asterisks indicate significant values (Pairwise t-test; rod b-wave P

    Journal: Scientific Reports

    Article Title: Disruption in murine Eml1 perturbs retinal lamination during early development

    doi: 10.1038/s41598-020-62373-5

    Figure Lengend Snippet: The laminar organization of the retina is perturbed in homozygous Eml1 tvrm360 eyes. ( a ) Retinal sections of control eyes show dense hematoxylin-stained nuclei in the outer nuclear layer (ONL) at postnatal day (P) 8 and 1 M. In contrast, in Eml1 tvrm360 mutant retinas dense hematoxylin-stained nuclei were also observed in the GCL at P8 and in the INL at P8 and 1 M. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 µm. ( b–g ) Full field ERG responses recorded in Eml1 tvrm360 (n = 5) and control littermates (n = 5) at one month of age. (b) Mean scotopic traces from a representative wild-type (WT, black ) and homozygous Eml1 tvrm360 littermate ( tvrm360 , blue) with increasing light stimulus intensities (values indicate flash illuminance in log cd s m-2). Scale bars: vertical, 500 µV; horizontal, 50 ms. Light dose response analysis showing mean ± SEM scotopic ( c ) b-wave and ( d ) a-wave amplitudes of WT (black circles) and tvrm360 (blue circles) mice. ( e ) Representative photopic traces from the same mice as in ( b ). Scale bars: vertical, 200 µV; horizontal, 50 ms. ( f ) Light dose response analysis showing mean ± standard error photopic b-wave amplitudes in WT (black circles) and tvrm360 (blue circles) mice. Asterisks indicate significant values (Pairwise t-test; rod b-wave P

    Article Snippet: To isolate the neural retina, eyes were removed and immediately placed in ice cold 1X PBS containing 10 mM Ribonucleoside Vanadyl Complex (S1402S, New England Biolabs).

    Techniques: Staining, Mutagenesis, Mouse Assay