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  • 94
    Neuromics nestin
    Impaired Development of Neural Progenitors in L3MBTL1-KD Pluripotent Stem Cells (A) KD of L3MBTL1 impairs the ability to form neuronal progenitor cells and neurons. <t>Nestin</t> and <t>Tuj1</t> expression were evaluated in EBs by IF. DAPI served as control. (Top) The first line shows multiple EBs at lesser magnification (4×); the second line shows a single EB at 10× magnification. (Bottom) The impairment in neural progenitor formation of the L3MBTL1-KD EBs. The graph on the right shows the percentage of EBs containing Tuj1 + cells. The data represent the mean ± SD of the three independent experiments. ∗ p
    Nestin, supplied by Neuromics, used in various techniques. Bioz Stars score: 94/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam nestin
    NSPC cultures. Immunohistochmistry confirmed that our NSPC cultures were positive for a NSPC marker <t>nestin</t> (A) and a proliferative marker <t>Ki67</t> (B). Nuclei were stained by DAPI shown in blue.
    Nestin, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank nestin
    Loss of <t>BAF53a</t> leads to reduced expression of cell cycle regulators and enhanced expression of differentiation genes. (a) Genome-wide gene expression changes measured by RNA-seq in BAF53a mutant ( <t>Nestin-Cre</t> ; Baf53a fl/– , KO) and heterozygous control ( Nestin-Cre ; Baf53a fl/+ , Het) forebrains at E15.5 (n=4). Magnitude and average (MA) plot (KO/Het) of RNA-seq data, displaying significantly changed genes in red (FDR
    Nestin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare nestin
    PD promoted neuronal‐like differentiation of BMSCs in the injured spinal cord. A, Immunofluorescence images of spinal cords showing neuron‐like cells in the PD + BMSC group (scale bar = 20 μm). Inset squares indicate the co‐localization of <t>BrdU</t> with <t>Nestin,</t> NeuN or NSE. B‐D, Quantitative analysis of fluorescence intensities, * P
    Nestin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech nestin
    Identification of Neural stem cells (NSCs) and microglia in vitro. Neurospheres isolated from ventral mesencephalic neural tissue were positive for <t>nestin</t> (A). After 7 days differentiation culture, the cells derived from NSCs were stained with antibodies against glial fibrillary acidic protein (GFAP) and <t>Tuj1</t> (B and C). Tyrosine hydroxylase (TH) immunoreactivity was detected in subpopulations of Tuj1+ neuronal cells (D). DAT expression was also detected in TH+ cells (E). Microglial cells immunopositive for CD11b (F). Scale bar: 20 μm
    Nestin, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems nestin
    Effects of miR-145 overexpression in vivo (A) Kaplan-Meier survival analysis shows that mice injected with miRover-NS survive longer than mice injected with Empty-NS (P = 0.003). (B) Immunohistochemistry of miRover and Empty tumors (40X). <t>Ki67-,</t> <t>Nestin-,</t> GFAP, DCX- and NEDD9-positive cell were counted on three to five independent 40X fields per tumor in 2-3 animals per group. miRover tumors present a higher amount of GFAP-positive cells and a lower amount of cells positive for Ki67, Nestin, DCX and NEDD9 compared to Empty. Histograms represent the quantification of positive cells: Ki67 (48 ± 1.2% in miRover vs. 53.2 ± 0.6 in empty), Nestin (6.2 ± 0.7% in miRover vs. 85.7 ± 1.3 in empty), GFAP (64.4 ± 1.1% in miRover vs. 3.5 ± 0.5 in empty), DCX (12.5 ± 1.7% in miRover vs. 34.5 ± 1.9 in empty), and NEDD9 (0 ± 0% in miRover vs. 89.3 ± 1.2 in empty). A representative image for each tumor is displayed (* P
    Nestin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology nestin
    Study of OPN immunoexpression in ENU-glioma stages of development There is an enhancement of the density of OPN+ cells corresponding to ENU-glioma malignant process. ( A ) In advanced stages II–III, these cells present different morphologies, as shown by DAB. Small round shape cells, cells of large soma and elongated cells contacting with a tumour microvessel is found within the neoplasia. ( B ) In the periphery area of stage III, many astrocyte-like cells tend to cluster. Confocal images of double immunofluorescence show positivity of astrocyte-like cell for OPN, <t>nestin</t> (green); VEGF, GFAP, and CD133 (red) (Hoechst counterstained, blue). Some of OPN+ cells and some of nestin+ cells co-express with GFAP, VEGF and CD133 (yellow). OPN expression is similar to CD133 but significantly lower than GFAP, VEGF and nestin one. Nestin is significantly lower than GFAP but similar to VEGF. Graphics show the <t>osteopontin</t> labelling index as mean percentage of positive cells (A) and quantitative study of the immunoexpression of the studied antibodies as mean percentage of border surface occupied by positive cells (B) ± typical error. * p
    Nestin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend nestin
    Abnormal <t>GFAP</t> expression in FMRP-deficient NPCs. (A) Representative immunofluorescence staining of the expression of SOX2 (red), <t>NESTIN</t> (gray) and GFAP (green) in iNPC-WT and iNPC-KO lines. Arrowheads indicate SOX2, NESTIN and GFAP-positive cells. Scale bars represent 100 μm. (B) Quantification of GFAP-positive cells in NPCs. The data are presented as mean ± standard deviation (S.D.) of three independent experiments (*** p
    Nestin, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore nestin
    NSCs characterization. The NSC was stained with <t>SOX1</t> (B) and <t>Nestin</t> antibodies (D). (A and C) are unstained corresponding images of (B and D). Over 90% of the NSCs were stained positive. The first three passage NSCs were harvested and intrinsic NSC markers, Pax6, Sox2, Nestin, EGFR and GFAP were tested by RT-PCR. Water was used as no loading control. The NSCs keep their NSC characteristic gene expression during passages.
    Nestin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4923 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies nestin
    NSCs characterization. The NSC was stained with <t>SOX1</t> (B) and <t>Nestin</t> antibodies (D). (A and C) are unstained corresponding images of (B and D). Over 90% of the NSCs were stained positive. The first three passage NSCs were harvested and intrinsic NSC markers, Pax6, Sox2, Nestin, EGFR and GFAP were tested by RT-PCR. Water was used as no loading control. The NSCs keep their NSC characteristic gene expression during passages.
    Nestin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AvesLabs nestin
    Human DPSCs grown in Neurocult proliferation share neural stem/progenitor markers with murine NSCs. (A,A’) Human DPSCs were allowed to grow for 7 days in laminin-coated wells in the presence of Neurocult proliferation media are <t>Nestin</t> positive and also express astroglial markers <t>GFAP</t> and S100ß ( n = 230). (B,B’) Murine NSCs grown for 3 days (to avoid confluency) in the same culture conditions also express these astroglial markers in similar proportion ( n = 242). Mean ± SEM of three independent experiments. Scale bar 75 μm.
    Nestin, supplied by AvesLabs, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson nestin
    MMP-2 and -9 were highly expressed on marginal region and tumor cells infiltrated the normal brain (arrow) in MTS23 group, while those in the control group were expressed on tumor, but not on normal region. And tumor cells were identified by immunohistochemistry for <t>Nestin.</t> MMP : <t>metalloproteinases.</t>
    Nestin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beyotime nestin
    Effects of IDH1-R132H on U87MG cell and U87MG-GSC radiosensitivity. (A) Expression of IDH1-R132H in U87MG cells transfected with the pCMV–IDH1-R132H plasmid was examined by western blot analysis. (B) The radiosensitivity of U87MG cells expressing IDH1-R132H was evaluated by clonogenic assay. (C) The differentiation marker <t>GFAP</t> and stem cell marker <t>nestin</t> were visualized in U87MG cells and U87MG-GSCs by immunofluorescence. (D) IDH1-R132H expression in U87MG-GSCs following transfection with the pCMV–IDH1-R132H plasmid. (E) Clonogenic assay of U87MG-GSCs transfected with an empty vector or the pCMV–IDH1-R132H plasmid. **P
    Nestin, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Pharmingen nestin
    ICAT –/– embryos lack the anterior neural plate. Whole-mount in situ hybridization of E8.0–E8.5 embryos was performed. ( A ) The anterior neural plate marker Six3 was barely detectable in ICAT –/– (right) embryos at E8.0 (1- to 2-somites stage). ( B ) In E8.0 wild-type embryos (left), Irx3 was expressed in the posterior neural plate, complementarily to Six3 expression in the anterior plate. In ICAT –/– (right) embryos, Irx3 was also expressed in the anterior neural plate (1- to 2-somites stage). ( C ) The anterior neural plate expressing BF-1 was barely detectable in ICAT –/– (right) embryos at E8.5 (8- to 10-somites stage). ( D ) The neural progenitor marker <t>Nestin</t> was similarly expressed in wild-type (left) and ICAT –/– (right) embryos at E8.5 (5- to 6-somites stage). ( E ) Neural progenitor cells expressing <t>Sox1</t> were normally induced in ICAT –/– embryos at E8.5 (right) (5- to 6-somites stage). ( F ) Expression of class III β-tubulin, a marker for differentiating neurons, at E8.5 appears normal in ICAT –/– embryos (right) (7- to 9-somites stage).
    Nestin, supplied by Pharmingen, used in various techniques. Bioz Stars score: 93/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    STEMCELL Technologies Inc nestin
    Immunophenotypic characterisation of iPSC differentiation following the conventional chemical induction with (ES+) and without (ES-) electrical stimulation. Expression of ( a ) early neuronal marker Tuj and neuroectodermal progenitor cell markers, ( b ) <t>nestin,</t> and ( c ) <t>Pax6.</t> Electrically stimulated cell cultures were distinguished by a higher antigen expression and diffuse nestin-labelled neurites outgrowths. Scale bars as indicated. ( d ) Quantitative assessment of Tuj1, nestin, and Pax6 immunocytochemistry (integrated density per cell; IntDen/Cell), confirming qualitative assessment. Mean ± S.D.; n = 3–5. One-way analysis of variance (ANOVA) with a Bonferroni post-hoc test. * p
    Nestin, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Spring Bioscience nestin
    Immunophenotypic characterisation of iPSC differentiation following the conventional chemical induction with (ES+) and without (ES-) electrical stimulation. Expression of ( a ) early neuronal marker Tuj and neuroectodermal progenitor cell markers, ( b ) <t>nestin,</t> and ( c ) <t>Pax6.</t> Electrically stimulated cell cultures were distinguished by a higher antigen expression and diffuse nestin-labelled neurites outgrowths. Scale bars as indicated. ( d ) Quantitative assessment of Tuj1, nestin, and Pax6 immunocytochemistry (integrated density per cell; IntDen/Cell), confirming qualitative assessment. Mean ± S.D.; n = 3–5. One-way analysis of variance (ANOVA) with a Bonferroni post-hoc test. * p
    Nestin, supplied by Spring Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Covance nestin
    Neuronal RIT1 expression increases HNPC proliferation and <t>Sox2</t> transcriptional activity. A and C , representative immunofluorescent images ( A ) and quantification ( C ) of the DG from DTG mice immunostained for <t>Nestin</t> ( green ) and Ki67 ( red ) to label proliferating neuronal stem cells. Nuclei (DAPI) are shown in blue. B , HNPCs were transfected with Myc-tagged RIT1 Q79L (CA) or EV, and proliferation was assessed by immunohistochemical detection of Nestin ( green ) and Ki67 ( red ) co-labeled cells. D , quantification of HNPC proliferation (Nestin + /Ki67 + ) as mean ± S.E. from three independent experiments. E , HNPCs were transfected as in A in the presence of a Sox2 luciferase reporter construct. Luciferase activity was evaluated 48 h post-transfection as described under “Experimental Procedures.” Results are presented as mean ± S.E. for three independent experiments repeated in triplicate. *, p
    Nestin, supplied by Covance, used in various techniques. Bioz Stars score: 93/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Neuromics chicken anti nestin
    β8 integrin is required for GSC self-renewal and differentiation in vitro . ( a ) Experimental approach for isolation of β8 high and β8 low primary GBM cells from human samples followed by in vitro culturing and/or intracranial injection. ( b ) Summary of β8 integrin protein expression levels as determined by FACS in 25 different freshly resected primary GBM samples. ( c, d ) β8 high GBM cells from sample HBT14 form spheroids and survive in culture ( c ), whereas β8 low cells do not form spheroids and fail to thrive in culture ( d ). Images shown are of spheroids formed from non-passaged β8 high and β8 low GBM cells. ( e ) Quantitation of β8 integrin-dependent sphere formation in vitro . ( f–i ) Spheroids generated from low-passage β8 high GBM cells (HBT41) were grown in the presence or absence of serum and immunofluorescently labeled with <t>anti-Nestin</t> and <t>anti-GFAP</t> to label neural stem cells and astrocytes ( f, g ) or anti-TUJ1 and anti-neurofilament antibodies to label neurons ( h, i ). ( j ) Neural stem cell markers were quantified by RT-PCR using spheroids generated from low-passage β8 high GBM cells (HBT28) before and after differentiation via serum exposure. ( k ) Serum-induced differentiation of β8 high GBM cells (HBT32) leads to reduced β8 integrin expression at low passages, but β8 integrin expression increases in more differentiated cells at higher passages.
    Chicken Anti Nestin, supplied by Neuromics, used in various techniques. Bioz Stars score: 89/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Miltenyi Biotec nestin
    Immunophenotyping of paediatric glioma cell lines. All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, <t>vimentin,</t> synaptophysin, <t>nestin</t> and CD133. H E – haematoxylin and eosin. All images original magnification ×400.
    Nestin, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer nestin
    Pax3 negatively regulates p53 protein, but not mRNA levels in ESC just as in mouse embryos. (A) Real time RT-PCR of p53 , Pax3 , and <t>Nestin</t> mRNA in stage 1 (open bars) and stage 3, days 2–8 (solid bars) ESC. Nestin mRNA is expressed in neuroepithelium and in ESC-derived neuronal precursors [29] , [30] and served as a control for a marker of neuroepithelial neuronal precursors. Each mRNA was normalized to <t>rRNA.</t> In (A), (B), and (D) values represent the mean ± SEM (n = 3 culture dishes). *p
    Nestin, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Impaired Development of Neural Progenitors in L3MBTL1-KD Pluripotent Stem Cells (A) KD of L3MBTL1 impairs the ability to form neuronal progenitor cells and neurons. Nestin and Tuj1 expression were evaluated in EBs by IF. DAPI served as control. (Top) The first line shows multiple EBs at lesser magnification (4×); the second line shows a single EB at 10× magnification. (Bottom) The impairment in neural progenitor formation of the L3MBTL1-KD EBs. The graph on the right shows the percentage of EBs containing Tuj1 + cells. The data represent the mean ± SD of the three independent experiments. ∗ p

    Journal: Stem Cell Reports

    Article Title: The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

    doi: 10.1016/j.stemcr.2015.02.003

    Figure Lengend Snippet: Impaired Development of Neural Progenitors in L3MBTL1-KD Pluripotent Stem Cells (A) KD of L3MBTL1 impairs the ability to form neuronal progenitor cells and neurons. Nestin and Tuj1 expression were evaluated in EBs by IF. DAPI served as control. (Top) The first line shows multiple EBs at lesser magnification (4×); the second line shows a single EB at 10× magnification. (Bottom) The impairment in neural progenitor formation of the L3MBTL1-KD EBs. The graph on the right shows the percentage of EBs containing Tuj1 + cells. The data represent the mean ± SD of the three independent experiments. ∗ p

    Article Snippet: The following primary antibodies were used: Tuj1 (Covance MRB435P-100), Nestin (Neuromics MO15012), and TAL1 (Abcam 119754).

    Techniques: Expressing

    Phenotypic profiling of astrocytes in the presence and absence of an ECM coating. Representative fluorescent images showing a heterogenous population of cells expressing nestin and GFAP at 15 DIV ( a ) and ~30 DIV ( b ). Scale bar = 50 μm. Bar graph summarizes flow cytometry data highlighting the distribution of astrocytic phenotypes (Nestin−GFAP−, Nestin+GFAP−, Nestin+GFAP+, Nestin−GFAP+) at 15 DIV ( c ) and ~30 DIV ( d ). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats).

    Journal: Scientific Reports

    Article Title: Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array

    doi: 10.1038/s41598-019-40128-1

    Figure Lengend Snippet: Phenotypic profiling of astrocytes in the presence and absence of an ECM coating. Representative fluorescent images showing a heterogenous population of cells expressing nestin and GFAP at 15 DIV ( a ) and ~30 DIV ( b ). Scale bar = 50 μm. Bar graph summarizes flow cytometry data highlighting the distribution of astrocytic phenotypes (Nestin−GFAP−, Nestin+GFAP−, Nestin+GFAP+, Nestin−GFAP+) at 15 DIV ( c ) and ~30 DIV ( d ). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats).

    Article Snippet: Cells were labeled with anti-class III beta-tubulin (Tuj1, chicken, 1:200, Neuromics, Edina, MN), anti-nestin (chicken, 1:200, Neuromics), anti-GFAP (mouse, 1:1000, Millipore-Sigma), anti-synaptophysin (rabbit, 1:250, Abcam, Cambridge, MA), or anti-Ki67 (mouse, 1:10, BD Biosciences).

    Techniques: Expressing, Flow Cytometry, Cytometry

    Characterization of cellular composition. Flow cytometry analysis of primary neuron and glial cells co-cultured in the presence or absence of an ECM coating. ( a ) Representative flow cytometry plots illustrate the gating strategy for the co-culture system: single cell population (top left) was gated based on forward-scatter characteristics, Zombie viability dye was used to exclude dead cells (high fluorescence) (top right), and Tuj1 staining was used to identify neuronal (Tuj1+) and glia (Tuj1−) subpopulation of cells (bottom left). The glial subpopulation (Tuj1− cells) was further gated to determine the phenotype of the astrocytic population based on nestin and GFAP expression (bottom right). Scatter plots summarizing the % of Tuj1+ ( b ) and % Tuj1− cells ( c ) from PDL, MaxGel, and bECM culture conditions across time (n = 2–3 cultures from 2 biological repeats). ( d ) Representative fluorescent images of DAPI-stained nuclei (blue) and those positive for the Ki67 proliferative marker (red). Scale bar = 50 μm. ( e ) Bar graph summarizes the total nuclei count (top) and % of Ki67 + nuclei (bottom). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats) indicated at the base of each bar and were analyzed using two-way ANOVA with Tukey’s post hoc test. Statistical significances between time points are at a level of ** p

    Journal: Scientific Reports

    Article Title: Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array

    doi: 10.1038/s41598-019-40128-1

    Figure Lengend Snippet: Characterization of cellular composition. Flow cytometry analysis of primary neuron and glial cells co-cultured in the presence or absence of an ECM coating. ( a ) Representative flow cytometry plots illustrate the gating strategy for the co-culture system: single cell population (top left) was gated based on forward-scatter characteristics, Zombie viability dye was used to exclude dead cells (high fluorescence) (top right), and Tuj1 staining was used to identify neuronal (Tuj1+) and glia (Tuj1−) subpopulation of cells (bottom left). The glial subpopulation (Tuj1− cells) was further gated to determine the phenotype of the astrocytic population based on nestin and GFAP expression (bottom right). Scatter plots summarizing the % of Tuj1+ ( b ) and % Tuj1− cells ( c ) from PDL, MaxGel, and bECM culture conditions across time (n = 2–3 cultures from 2 biological repeats). ( d ) Representative fluorescent images of DAPI-stained nuclei (blue) and those positive for the Ki67 proliferative marker (red). Scale bar = 50 μm. ( e ) Bar graph summarizes the total nuclei count (top) and % of Ki67 + nuclei (bottom). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats) indicated at the base of each bar and were analyzed using two-way ANOVA with Tukey’s post hoc test. Statistical significances between time points are at a level of ** p

    Article Snippet: Cells were labeled with anti-class III beta-tubulin (Tuj1, chicken, 1:200, Neuromics, Edina, MN), anti-nestin (chicken, 1:200, Neuromics), anti-GFAP (mouse, 1:1000, Millipore-Sigma), anti-synaptophysin (rabbit, 1:250, Abcam, Cambridge, MA), or anti-Ki67 (mouse, 1:10, BD Biosciences).

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Co-Culture Assay, Fluorescence, Staining, Expressing, Marker

    NSPC cultures. Immunohistochmistry confirmed that our NSPC cultures were positive for a NSPC marker nestin (A) and a proliferative marker Ki67 (B). Nuclei were stained by DAPI shown in blue.

    Journal: PLoS ONE

    Article Title: Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells

    doi: 10.1371/journal.pone.0138724

    Figure Lengend Snippet: NSPC cultures. Immunohistochmistry confirmed that our NSPC cultures were positive for a NSPC marker nestin (A) and a proliferative marker Ki67 (B). Nuclei were stained by DAPI shown in blue.

    Article Snippet: Immunocytochemistry To confirm the status/purity of primary cultured NSPCs, the cells were stained with nestin (1:100, Abcam, Cambridge, MA, USA) and Ki67 (1:100, Abcam) antibodies, which are well-known markers for NSPCs [ , ].

    Techniques: Marker, Staining

    Loss of BAF53a leads to reduced expression of cell cycle regulators and enhanced expression of differentiation genes. (a) Genome-wide gene expression changes measured by RNA-seq in BAF53a mutant ( Nestin-Cre ; Baf53a fl/– , KO) and heterozygous control ( Nestin-Cre ; Baf53a fl/+ , Het) forebrains at E15.5 (n=4). Magnitude and average (MA) plot (KO/Het) of RNA-seq data, displaying significantly changed genes in red (FDR

    Journal: bioRxiv

    Article Title: The npBAF to nBAF Chromatin Switch Regulates Cell Cycle Exit in the Developing Mammalian Cortex

    doi: 10.1101/2020.01.17.910794

    Figure Lengend Snippet: Loss of BAF53a leads to reduced expression of cell cycle regulators and enhanced expression of differentiation genes. (a) Genome-wide gene expression changes measured by RNA-seq in BAF53a mutant ( Nestin-Cre ; Baf53a fl/– , KO) and heterozygous control ( Nestin-Cre ; Baf53a fl/+ , Het) forebrains at E15.5 (n=4). Magnitude and average (MA) plot (KO/Het) of RNA-seq data, displaying significantly changed genes in red (FDR

    Article Snippet: Antibodies used were Nestin (DSHB; Rat-401), Map2ab (Millipore, MAB378Tuj1), BAF53a (Novus, NB100-61628), BAF53b (Crabtree lab).

    Techniques: Expressing, Genome Wide, RNA Sequencing Assay, Mutagenesis

    BAF53a regulates chromatin accessibility at NPC identity and neural differentiation genes. (a) Genome-wide chromatin accessibility changes as measured by ATAC-seq in BAF53a mutant ( Nestin-Cre ; Baf53a fl/– , KO) and heterozygous control ( Nestin-Cre ; Baf53a fl/+ , Het) forebrains at E15.5 (n=4). Magnitude and average (MA) plot (KO/Het) of ATAC-seq data, displaying significantly changed peaks in red (FDR

    Journal: bioRxiv

    Article Title: The npBAF to nBAF Chromatin Switch Regulates Cell Cycle Exit in the Developing Mammalian Cortex

    doi: 10.1101/2020.01.17.910794

    Figure Lengend Snippet: BAF53a regulates chromatin accessibility at NPC identity and neural differentiation genes. (a) Genome-wide chromatin accessibility changes as measured by ATAC-seq in BAF53a mutant ( Nestin-Cre ; Baf53a fl/– , KO) and heterozygous control ( Nestin-Cre ; Baf53a fl/+ , Het) forebrains at E15.5 (n=4). Magnitude and average (MA) plot (KO/Het) of ATAC-seq data, displaying significantly changed peaks in red (FDR

    Article Snippet: Antibodies used were Nestin (DSHB; Rat-401), Map2ab (Millipore, MAB378Tuj1), BAF53a (Novus, NB100-61628), BAF53b (Crabtree lab).

    Techniques: Genome Wide, Mutagenesis

    PD promoted neuronal‐like differentiation of BMSCs in the injured spinal cord. A, Immunofluorescence images of spinal cords showing neuron‐like cells in the PD + BMSC group (scale bar = 20 μm). Inset squares indicate the co‐localization of BrdU with Nestin, NeuN or NSE. B‐D, Quantitative analysis of fluorescence intensities, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway, et al. Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway

    doi: 10.1111/jcmm.15187

    Figure Lengend Snippet: PD promoted neuronal‐like differentiation of BMSCs in the injured spinal cord. A, Immunofluorescence images of spinal cords showing neuron‐like cells in the PD + BMSC group (scale bar = 20 μm). Inset squares indicate the co‐localization of BrdU with Nestin, NeuN or NSE. B‐D, Quantitative analysis of fluorescence intensities, * P

    Article Snippet: For immunofluorescence, 10‐μm‐thick transverse frozen sections were incubated with primary antibodies targeting MAP‐2 (1:200) and Nrf2 (1:200, R & D systems), and the longitudinal sections, with antibodies against GFAP (1:500, Abcam), NF‐200 (1:200, Boster), Tuj1 (1:200, CST), GAP43 (1:300, Novus), BrdU (1:200), Nestin (1:200, Novus), NeuN (1:300) and NSE (1:100, Abcam).

    Techniques: Immunofluorescence, Fluorescence

    Identification of Neural stem cells (NSCs) and microglia in vitro. Neurospheres isolated from ventral mesencephalic neural tissue were positive for nestin (A). After 7 days differentiation culture, the cells derived from NSCs were stained with antibodies against glial fibrillary acidic protein (GFAP) and Tuj1 (B and C). Tyrosine hydroxylase (TH) immunoreactivity was detected in subpopulations of Tuj1+ neuronal cells (D). DAT expression was also detected in TH+ cells (E). Microglial cells immunopositive for CD11b (F). Scale bar: 20 μm

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Transplantation of Nurr1‐overexpressing neural stem cells and microglia for treating parkinsonian rats, et al. Transplantation of Nurr1‐overexpressing neural stem cells and microglia for treating parkinsonian rats

    doi: 10.1111/cns.13149

    Figure Lengend Snippet: Identification of Neural stem cells (NSCs) and microglia in vitro. Neurospheres isolated from ventral mesencephalic neural tissue were positive for nestin (A). After 7 days differentiation culture, the cells derived from NSCs were stained with antibodies against glial fibrillary acidic protein (GFAP) and Tuj1 (B and C). Tyrosine hydroxylase (TH) immunoreactivity was detected in subpopulations of Tuj1+ neuronal cells (D). DAT expression was also detected in TH+ cells (E). Microglial cells immunopositive for CD11b (F). Scale bar: 20 μm

    Article Snippet: Cultured cells or cryosectioned brain slices were fixed with 4% paraformaldehyde in PBS and incubated overnight at 4°C with the following primary antibodies: Nestin (1:500, rabbit, Proteintech); Tuj1 (1:500, mouse, Abcam, MA, USA); TH (1:250, rabbit, Abcam); TH (1:500, mouse, Proteintech); DA transporter (DAT, 1:500, rabbit, Abcam); CD11b/c (1:500, mouse, Abcam); and Iba‐I (1:250, rabbit, Abcam).

    Techniques: In Vitro, Isolation, Derivative Assay, Staining, Expressing

    Effects of miR-145 overexpression in vivo (A) Kaplan-Meier survival analysis shows that mice injected with miRover-NS survive longer than mice injected with Empty-NS (P = 0.003). (B) Immunohistochemistry of miRover and Empty tumors (40X). Ki67-, Nestin-, GFAP, DCX- and NEDD9-positive cell were counted on three to five independent 40X fields per tumor in 2-3 animals per group. miRover tumors present a higher amount of GFAP-positive cells and a lower amount of cells positive for Ki67, Nestin, DCX and NEDD9 compared to Empty. Histograms represent the quantification of positive cells: Ki67 (48 ± 1.2% in miRover vs. 53.2 ± 0.6 in empty), Nestin (6.2 ± 0.7% in miRover vs. 85.7 ± 1.3 in empty), GFAP (64.4 ± 1.1% in miRover vs. 3.5 ± 0.5 in empty), DCX (12.5 ± 1.7% in miRover vs. 34.5 ± 1.9 in empty), and NEDD9 (0 ± 0% in miRover vs. 89.3 ± 1.2 in empty). A representative image for each tumor is displayed (* P

    Journal: Oncotarget

    Article Title: NEDD9, a novel target of miR-145, increases the invasiveness of glioblastoma

    doi:

    Figure Lengend Snippet: Effects of miR-145 overexpression in vivo (A) Kaplan-Meier survival analysis shows that mice injected with miRover-NS survive longer than mice injected with Empty-NS (P = 0.003). (B) Immunohistochemistry of miRover and Empty tumors (40X). Ki67-, Nestin-, GFAP, DCX- and NEDD9-positive cell were counted on three to five independent 40X fields per tumor in 2-3 animals per group. miRover tumors present a higher amount of GFAP-positive cells and a lower amount of cells positive for Ki67, Nestin, DCX and NEDD9 compared to Empty. Histograms represent the quantification of positive cells: Ki67 (48 ± 1.2% in miRover vs. 53.2 ± 0.6 in empty), Nestin (6.2 ± 0.7% in miRover vs. 85.7 ± 1.3 in empty), GFAP (64.4 ± 1.1% in miRover vs. 3.5 ± 0.5 in empty), DCX (12.5 ± 1.7% in miRover vs. 34.5 ± 1.9 in empty), and NEDD9 (0 ± 0% in miRover vs. 89.3 ± 1.2 in empty). A representative image for each tumor is displayed (* P

    Article Snippet: Immunohistochemistry and immunofluorescence analyses Immunohistochemical analyses for Ki67 (1:100, BD, New Jersey, USA), Nestin (1:100, R & D System, Minnesota, USA), GFAP (1:100, DAKO, Glostrup, Denmark), DXC (Abcam, 1:100) and NEDD9 (Acris, 1:50) were performed on paraffin-embedded sections.

    Techniques: Over Expression, In Vivo, Mouse Assay, Injection, Immunohistochemistry

    Study of OPN immunoexpression in ENU-glioma stages of development There is an enhancement of the density of OPN+ cells corresponding to ENU-glioma malignant process. ( A ) In advanced stages II–III, these cells present different morphologies, as shown by DAB. Small round shape cells, cells of large soma and elongated cells contacting with a tumour microvessel is found within the neoplasia. ( B ) In the periphery area of stage III, many astrocyte-like cells tend to cluster. Confocal images of double immunofluorescence show positivity of astrocyte-like cell for OPN, nestin (green); VEGF, GFAP, and CD133 (red) (Hoechst counterstained, blue). Some of OPN+ cells and some of nestin+ cells co-express with GFAP, VEGF and CD133 (yellow). OPN expression is similar to CD133 but significantly lower than GFAP, VEGF and nestin one. Nestin is significantly lower than GFAP but similar to VEGF. Graphics show the osteopontin labelling index as mean percentage of positive cells (A) and quantitative study of the immunoexpression of the studied antibodies as mean percentage of border surface occupied by positive cells (B) ± typical error. * p

    Journal: Oncotarget

    Article Title: Association of Notch-1, osteopontin and stem-like cells in ENU-glioma malignant process

    doi: 10.18632/oncotarget.25808

    Figure Lengend Snippet: Study of OPN immunoexpression in ENU-glioma stages of development There is an enhancement of the density of OPN+ cells corresponding to ENU-glioma malignant process. ( A ) In advanced stages II–III, these cells present different morphologies, as shown by DAB. Small round shape cells, cells of large soma and elongated cells contacting with a tumour microvessel is found within the neoplasia. ( B ) In the periphery area of stage III, many astrocyte-like cells tend to cluster. Confocal images of double immunofluorescence show positivity of astrocyte-like cell for OPN, nestin (green); VEGF, GFAP, and CD133 (red) (Hoechst counterstained, blue). Some of OPN+ cells and some of nestin+ cells co-express with GFAP, VEGF and CD133 (yellow). OPN expression is similar to CD133 but significantly lower than GFAP, VEGF and nestin one. Nestin is significantly lower than GFAP but similar to VEGF. Graphics show the osteopontin labelling index as mean percentage of positive cells (A) and quantitative study of the immunoexpression of the studied antibodies as mean percentage of border surface occupied by positive cells (B) ± typical error. * p

    Article Snippet: Primary monoclonal antibodies used were nestin (1:400; sc-21247, Santa Cruz, CA) and osteopontin (OPN, 1:300; sc-21742, Santa Cruz, CA).

    Techniques: Immunofluorescence, Expressing

    Abnormal GFAP expression in FMRP-deficient NPCs. (A) Representative immunofluorescence staining of the expression of SOX2 (red), NESTIN (gray) and GFAP (green) in iNPC-WT and iNPC-KO lines. Arrowheads indicate SOX2, NESTIN and GFAP-positive cells. Scale bars represent 100 μm. (B) Quantification of GFAP-positive cells in NPCs. The data are presented as mean ± standard deviation (S.D.) of three independent experiments (*** p

    Journal: Scientific Reports

    Article Title: Loss of the fragile X mental retardation protein causes aberrant differentiation in human neural progenitor cells

    doi: 10.1038/s41598-018-30025-4

    Figure Lengend Snippet: Abnormal GFAP expression in FMRP-deficient NPCs. (A) Representative immunofluorescence staining of the expression of SOX2 (red), NESTIN (gray) and GFAP (green) in iNPC-WT and iNPC-KO lines. Arrowheads indicate SOX2, NESTIN and GFAP-positive cells. Scale bars represent 100 μm. (B) Quantification of GFAP-positive cells in NPCs. The data are presented as mean ± standard deviation (S.D.) of three independent experiments (*** p

    Article Snippet: The first antibodies used were directed against SSEA-4 (1/500, #MAB4304, Merck), GFAP (1/1000, #130300, Thermo Fisher Scientific), NESTIN (1/200, #MAB5326, Merck), NESTIN (1/200, #PRB570c-100, BioLegend, San Diego, CA, USA) SOX2 (1/500, #PM059, MBL, Nagoya, Japan), VGLUT1 (1/2000, #135303, Synaptic Systems, Göttingen, Germany) and MAP2 (1/5000, #ab5392, Abcam).

    Techniques: Expressing, Immunofluorescence, Staining, Standard Deviation

    NSCs characterization. The NSC was stained with SOX1 (B) and Nestin antibodies (D). (A and C) are unstained corresponding images of (B and D). Over 90% of the NSCs were stained positive. The first three passage NSCs were harvested and intrinsic NSC markers, Pax6, Sox2, Nestin, EGFR and GFAP were tested by RT-PCR. Water was used as no loading control. The NSCs keep their NSC characteristic gene expression during passages.

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: A novel method to derive and expand mice neural stem cells efficiently without neuro-sphere formation

    doi:

    Figure Lengend Snippet: NSCs characterization. The NSC was stained with SOX1 (B) and Nestin antibodies (D). (A and C) are unstained corresponding images of (B and D). Over 90% of the NSCs were stained positive. The first three passage NSCs were harvested and intrinsic NSC markers, Pax6, Sox2, Nestin, EGFR and GFAP were tested by RT-PCR. Water was used as no loading control. The NSCs keep their NSC characteristic gene expression during passages.

    Article Snippet: The following proteins were evaluated: Nestin (mouse monoclonal, 1:200, Chemicon, 2C13B9); Sox1 (rabbit polyclonal, 1:200, Chemicon, AB5768); b-III-tubulin (TuJ-1, mouse monoclonal, 1:200, Chemicon, MAB1637); glial fibrillary acid protein (GFAP, 1:400, Sigma, SAB1405864); Synapsin (1:500, Sigma, SAB4502905).

    Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

    Human DPSCs grown in Neurocult proliferation share neural stem/progenitor markers with murine NSCs. (A,A’) Human DPSCs were allowed to grow for 7 days in laminin-coated wells in the presence of Neurocult proliferation media are Nestin positive and also express astroglial markers GFAP and S100ß ( n = 230). (B,B’) Murine NSCs grown for 3 days (to avoid confluency) in the same culture conditions also express these astroglial markers in similar proportion ( n = 242). Mean ± SEM of three independent experiments. Scale bar 75 μm.

    Journal: Frontiers in Physiology

    Article Title: Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain

    doi: 10.3389/fphys.2019.00347

    Figure Lengend Snippet: Human DPSCs grown in Neurocult proliferation share neural stem/progenitor markers with murine NSCs. (A,A’) Human DPSCs were allowed to grow for 7 days in laminin-coated wells in the presence of Neurocult proliferation media are Nestin positive and also express astroglial markers GFAP and S100ß ( n = 230). (B,B’) Murine NSCs grown for 3 days (to avoid confluency) in the same culture conditions also express these astroglial markers in similar proportion ( n = 242). Mean ± SEM of three independent experiments. Scale bar 75 μm.

    Article Snippet: They were then incubated overnight at 4°C with primary antibodies at the following dilutions: glial fibrillary acidic protein (GFAP) (MAB3402, 1:400; Millipore, Lake Placid, NY, United States), Nestin (NES, 1:200 Aves Labs), S100ß (Z0311, 1:1000, Dako, Glostrup, Denmark), NeuN (EPR12763, 1:200; Abcam, Cambridge, United Kingdom), doublecortin (DCX) (sc-8066, 1:200; Santa Cruz, Dallas, TX, United States), CD31 (550247, 1:300 BD Pharmingen, San Jose, CA, United States), anti-VEGFR (#9698S 1:800, Cell Signaling Technologies, Danvers, MA, United States) anti-STAT3 (phospho Y705) antibody (ab76315, 1:500 Abcam), anti-human-CD31 (F8402, 1:200, Sigma St. Louis, MO, United States).

    Techniques:

    MMP-2 and -9 were highly expressed on marginal region and tumor cells infiltrated the normal brain (arrow) in MTS23 group, while those in the control group were expressed on tumor, but not on normal region. And tumor cells were identified by immunohistochemistry for Nestin. MMP : metalloproteinases.

    Journal: Journal of Korean Neurosurgical Society

    Article Title: Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model

    doi: 10.3340/jkns.2016.59.6.551

    Figure Lengend Snippet: MMP-2 and -9 were highly expressed on marginal region and tumor cells infiltrated the normal brain (arrow) in MTS23 group, while those in the control group were expressed on tumor, but not on normal region. And tumor cells were identified by immunohistochemistry for Nestin. MMP : metalloproteinases.

    Article Snippet: The following primary monoclonal antibodies were added : matrix metalloproteinases-2 and MMP-9 (1 : 8000; Abcam, Cambridge, UK) and Nestin (1 : 4000, BD Biosciences PharMingen, Burlingame, CA, USA).

    Techniques: Immunohistochemistry

    Effects of IDH1-R132H on U87MG cell and U87MG-GSC radiosensitivity. (A) Expression of IDH1-R132H in U87MG cells transfected with the pCMV–IDH1-R132H plasmid was examined by western blot analysis. (B) The radiosensitivity of U87MG cells expressing IDH1-R132H was evaluated by clonogenic assay. (C) The differentiation marker GFAP and stem cell marker nestin were visualized in U87MG cells and U87MG-GSCs by immunofluorescence. (D) IDH1-R132H expression in U87MG-GSCs following transfection with the pCMV–IDH1-R132H plasmid. (E) Clonogenic assay of U87MG-GSCs transfected with an empty vector or the pCMV–IDH1-R132H plasmid. **P

    Journal: Oncology Letters

    Article Title: IDH1-R132H mutation radiosensitizes U87MG glioma cells via epigenetic downregulation of TIGAR

    doi: 10.3892/ol.2019.11148

    Figure Lengend Snippet: Effects of IDH1-R132H on U87MG cell and U87MG-GSC radiosensitivity. (A) Expression of IDH1-R132H in U87MG cells transfected with the pCMV–IDH1-R132H plasmid was examined by western blot analysis. (B) The radiosensitivity of U87MG cells expressing IDH1-R132H was evaluated by clonogenic assay. (C) The differentiation marker GFAP and stem cell marker nestin were visualized in U87MG cells and U87MG-GSCs by immunofluorescence. (D) IDH1-R132H expression in U87MG-GSCs following transfection with the pCMV–IDH1-R132H plasmid. (E) Clonogenic assay of U87MG-GSCs transfected with an empty vector or the pCMV–IDH1-R132H plasmid. **P

    Article Snippet: Immunostaining was performed using antibodies against glial fibrillary acidic protein (GFAP, 1:500 Abcam; cat. no. ab7260) or nestin (1:100, Beyotime; AN203) at 4°C overnight.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Clonogenic Assay, Marker, Immunofluorescence

    ICAT –/– embryos lack the anterior neural plate. Whole-mount in situ hybridization of E8.0–E8.5 embryos was performed. ( A ) The anterior neural plate marker Six3 was barely detectable in ICAT –/– (right) embryos at E8.0 (1- to 2-somites stage). ( B ) In E8.0 wild-type embryos (left), Irx3 was expressed in the posterior neural plate, complementarily to Six3 expression in the anterior plate. In ICAT –/– (right) embryos, Irx3 was also expressed in the anterior neural plate (1- to 2-somites stage). ( C ) The anterior neural plate expressing BF-1 was barely detectable in ICAT –/– (right) embryos at E8.5 (8- to 10-somites stage). ( D ) The neural progenitor marker Nestin was similarly expressed in wild-type (left) and ICAT –/– (right) embryos at E8.5 (5- to 6-somites stage). ( E ) Neural progenitor cells expressing Sox1 were normally induced in ICAT –/– embryos at E8.5 (right) (5- to 6-somites stage). ( F ) Expression of class III β-tubulin, a marker for differentiating neurons, at E8.5 appears normal in ICAT –/– embryos (right) (7- to 9-somites stage).

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

    Article Title: Anteriorization of neural fate by inhibitor of ?-catenin and T cell factor (ICAT), a negative regulator of Wnt signaling

    doi: 10.1073/pnas.0401733101

    Figure Lengend Snippet: ICAT –/– embryos lack the anterior neural plate. Whole-mount in situ hybridization of E8.0–E8.5 embryos was performed. ( A ) The anterior neural plate marker Six3 was barely detectable in ICAT –/– (right) embryos at E8.0 (1- to 2-somites stage). ( B ) In E8.0 wild-type embryos (left), Irx3 was expressed in the posterior neural plate, complementarily to Six3 expression in the anterior plate. In ICAT –/– (right) embryos, Irx3 was also expressed in the anterior neural plate (1- to 2-somites stage). ( C ) The anterior neural plate expressing BF-1 was barely detectable in ICAT –/– (right) embryos at E8.5 (8- to 10-somites stage). ( D ) The neural progenitor marker Nestin was similarly expressed in wild-type (left) and ICAT –/– (right) embryos at E8.5 (5- to 6-somites stage). ( E ) Neural progenitor cells expressing Sox1 were normally induced in ICAT –/– embryos at E8.5 (right) (5- to 6-somites stage). ( F ) Expression of class III β-tubulin, a marker for differentiating neurons, at E8.5 appears normal in ICAT –/– embryos (right) (7- to 9-somites stage).

    Article Snippet: Dilution of antibodies was as follows: mAb to Nestin (Pharmingen), 1:500; polyclonal antibody (pAb) to Sox1 (sex-determining region Y box 1; provided by R. Lovell-Badge, National Institute for Medical Research, London; ref. ), 1:500; mAb to Otx1 (orthodenticle homolog 1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City), 1:1; mAb to Hoxc8 (homeobox C8; Covance, Princeton), 1:100.

    Techniques: In Situ Hybridization, Marker, Expressing

    Wnt signaling changes the fate of neural progenitors that differentiated from ES cells into a posterior character. ( A ) Semiquantitative RT-PCR analysis of Wnt3a-treated ES cells. Lane 1, undifferentiated ES cells. Lanes 2 and 3, SDIA-treated ES cells without or with Wnt3a treatment during days 2–6. Total RNA was isolated on day 6. Wnt3a treatment suppressed the forebrain markers Six3, BF-1, Emx2, Otx1, and Otx2 and induced the hindbrain markers Gbx2 and Hoxb1 and the spinal cord markers Irx3, Hoxb4, and Hoxc8. The levels of Nestin and Sox1 expression were not significantly affected. ( B – S ) Double immunostaining of SDIA-treated ES cells without ( B – D , H – J , and N – P ) or with ( E – G , K – M , and Q – S ) Wnt3a treatment during days 2–6. The day on which ES cells were seeded on PA6 was designated day 0. ( B – G ) Cells were double-stained with anti-Nestin and anti-Sox1 antibodies. ( H – M ) Cells were double-stained with anti-Otx1 and anti-Sox1 antibodies. ( N – S ) Cells were double-stained with anti-Hoxc8 and anti-Sox1 antibodies. The nuclei were stained with TO-PRO-3. Wnt3a treatment decreased Otx1-positive cells ( K ) and induced Hoxc8-positive cells ( Q ), whereas the levels of Nestin and Sox1 expression were not significantly affected. Otx1- or Hoxc8-positive cells also expressed Sox1, suggesting that these cells are neural progenitors.

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

    Article Title: Anteriorization of neural fate by inhibitor of ?-catenin and T cell factor (ICAT), a negative regulator of Wnt signaling

    doi: 10.1073/pnas.0401733101

    Figure Lengend Snippet: Wnt signaling changes the fate of neural progenitors that differentiated from ES cells into a posterior character. ( A ) Semiquantitative RT-PCR analysis of Wnt3a-treated ES cells. Lane 1, undifferentiated ES cells. Lanes 2 and 3, SDIA-treated ES cells without or with Wnt3a treatment during days 2–6. Total RNA was isolated on day 6. Wnt3a treatment suppressed the forebrain markers Six3, BF-1, Emx2, Otx1, and Otx2 and induced the hindbrain markers Gbx2 and Hoxb1 and the spinal cord markers Irx3, Hoxb4, and Hoxc8. The levels of Nestin and Sox1 expression were not significantly affected. ( B – S ) Double immunostaining of SDIA-treated ES cells without ( B – D , H – J , and N – P ) or with ( E – G , K – M , and Q – S ) Wnt3a treatment during days 2–6. The day on which ES cells were seeded on PA6 was designated day 0. ( B – G ) Cells were double-stained with anti-Nestin and anti-Sox1 antibodies. ( H – M ) Cells were double-stained with anti-Otx1 and anti-Sox1 antibodies. ( N – S ) Cells were double-stained with anti-Hoxc8 and anti-Sox1 antibodies. The nuclei were stained with TO-PRO-3. Wnt3a treatment decreased Otx1-positive cells ( K ) and induced Hoxc8-positive cells ( Q ), whereas the levels of Nestin and Sox1 expression were not significantly affected. Otx1- or Hoxc8-positive cells also expressed Sox1, suggesting that these cells are neural progenitors.

    Article Snippet: Dilution of antibodies was as follows: mAb to Nestin (Pharmingen), 1:500; polyclonal antibody (pAb) to Sox1 (sex-determining region Y box 1; provided by R. Lovell-Badge, National Institute for Medical Research, London; ref. ), 1:500; mAb to Otx1 (orthodenticle homolog 1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City), 1:1; mAb to Hoxc8 (homeobox C8; Covance, Princeton), 1:100.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Double Immunostaining, Staining

    Immunophenotypic characterisation of iPSC differentiation following the conventional chemical induction with (ES+) and without (ES-) electrical stimulation. Expression of ( a ) early neuronal marker Tuj and neuroectodermal progenitor cell markers, ( b ) nestin, and ( c ) Pax6. Electrically stimulated cell cultures were distinguished by a higher antigen expression and diffuse nestin-labelled neurites outgrowths. Scale bars as indicated. ( d ) Quantitative assessment of Tuj1, nestin, and Pax6 immunocytochemistry (integrated density per cell; IntDen/Cell), confirming qualitative assessment. Mean ± S.D.; n = 3–5. One-way analysis of variance (ANOVA) with a Bonferroni post-hoc test. * p

    Journal: Cells

    Article Title: Conducting Polymer Mediated Electrical Stimulation Induces Multilineage Differentiation with Robust Neuronal Fate Determination of Human Induced Pluripotent Stem Cells

    doi: 10.3390/cells9030658

    Figure Lengend Snippet: Immunophenotypic characterisation of iPSC differentiation following the conventional chemical induction with (ES+) and without (ES-) electrical stimulation. Expression of ( a ) early neuronal marker Tuj and neuroectodermal progenitor cell markers, ( b ) nestin, and ( c ) Pax6. Electrically stimulated cell cultures were distinguished by a higher antigen expression and diffuse nestin-labelled neurites outgrowths. Scale bars as indicated. ( d ) Quantitative assessment of Tuj1, nestin, and Pax6 immunocytochemistry (integrated density per cell; IntDen/Cell), confirming qualitative assessment. Mean ± S.D.; n = 3–5. One-way analysis of variance (ANOVA) with a Bonferroni post-hoc test. * p

    Article Snippet: The samples were subsequently incubated with primary antibodies in a blocking buffer for octamer-binding transcription factor 4 (OCT4; mouse; STEMCELL Technologies), sex determining region Y-box 2 (SOX2; rabbit; Millipore, Temecula, CA, USA), stage-specific embryonic antigen 4 (SSEA4; mouse; STEMCELL Technologies), neuron-specific class III β-tubulin (Tuj1; mouse; Covance, Princeton, NJ, USA), and neuroectodermal progenitor cell markers Vimentin (chicken; Millipore, Temecula, CA, USA), Pax6 (rabbit; Sigma Aldrich), and nestin (mouse; STEMCELL Technologies), or Alexa Fluor® conjugated antibodies for Tuj1 (mouse; BD Biosciences, San Jose, CA, USA), glial fibrillary acidic protein (GFAP; mouse; BD Biosciences) and nestin (mouse; BD Biosciences) at 4 °C overnight.

    Techniques: Expressing, Marker, Immunocytochemistry

    Immunophenotypic characterisation of iPSC differentiability: expression of neuroectodermal progenitor cell markers Pax6, nestin, and vimentin, early neuronal marker Tuj, and glial cell marker GFAP at ( a ) day 6, ( b ) day 12, or ( c ) day 18 of iPSC differentiation. Day 18 cell cultures were distinguished by more prominent neurite outgrowths emanating from Tuj1-expressing neurons. GFAP-expressing cells frequently appeared contigous with neurons, signified by two adjacent DAPI-labelled nuclei (arrows) and converged fluorescent labelling of adjoining glia and neurons. Scale bars as indicated.

    Journal: Cells

    Article Title: Conducting Polymer Mediated Electrical Stimulation Induces Multilineage Differentiation with Robust Neuronal Fate Determination of Human Induced Pluripotent Stem Cells

    doi: 10.3390/cells9030658

    Figure Lengend Snippet: Immunophenotypic characterisation of iPSC differentiability: expression of neuroectodermal progenitor cell markers Pax6, nestin, and vimentin, early neuronal marker Tuj, and glial cell marker GFAP at ( a ) day 6, ( b ) day 12, or ( c ) day 18 of iPSC differentiation. Day 18 cell cultures were distinguished by more prominent neurite outgrowths emanating from Tuj1-expressing neurons. GFAP-expressing cells frequently appeared contigous with neurons, signified by two adjacent DAPI-labelled nuclei (arrows) and converged fluorescent labelling of adjoining glia and neurons. Scale bars as indicated.

    Article Snippet: The samples were subsequently incubated with primary antibodies in a blocking buffer for octamer-binding transcription factor 4 (OCT4; mouse; STEMCELL Technologies), sex determining region Y-box 2 (SOX2; rabbit; Millipore, Temecula, CA, USA), stage-specific embryonic antigen 4 (SSEA4; mouse; STEMCELL Technologies), neuron-specific class III β-tubulin (Tuj1; mouse; Covance, Princeton, NJ, USA), and neuroectodermal progenitor cell markers Vimentin (chicken; Millipore, Temecula, CA, USA), Pax6 (rabbit; Sigma Aldrich), and nestin (mouse; STEMCELL Technologies), or Alexa Fluor® conjugated antibodies for Tuj1 (mouse; BD Biosciences, San Jose, CA, USA), glial fibrillary acidic protein (GFAP; mouse; BD Biosciences) and nestin (mouse; BD Biosciences) at 4 °C overnight.

    Techniques: Expressing, Marker

    Neuronal RIT1 expression increases HNPC proliferation and Sox2 transcriptional activity. A and C , representative immunofluorescent images ( A ) and quantification ( C ) of the DG from DTG mice immunostained for Nestin ( green ) and Ki67 ( red ) to label proliferating neuronal stem cells. Nuclei (DAPI) are shown in blue. B , HNPCs were transfected with Myc-tagged RIT1 Q79L (CA) or EV, and proliferation was assessed by immunohistochemical detection of Nestin ( green ) and Ki67 ( red ) co-labeled cells. D , quantification of HNPC proliferation (Nestin + /Ki67 + ) as mean ± S.E. from three independent experiments. E , HNPCs were transfected as in A in the presence of a Sox2 luciferase reporter construct. Luciferase activity was evaluated 48 h post-transfection as described under “Experimental Procedures.” Results are presented as mean ± S.E. for three independent experiments repeated in triplicate. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: RIT1 GTPase Regulates Sox2 Transcriptional Activity and Hippocampal Neurogenesis *

    doi: 10.1074/jbc.M116.749770

    Figure Lengend Snippet: Neuronal RIT1 expression increases HNPC proliferation and Sox2 transcriptional activity. A and C , representative immunofluorescent images ( A ) and quantification ( C ) of the DG from DTG mice immunostained for Nestin ( green ) and Ki67 ( red ) to label proliferating neuronal stem cells. Nuclei (DAPI) are shown in blue. B , HNPCs were transfected with Myc-tagged RIT1 Q79L (CA) or EV, and proliferation was assessed by immunohistochemical detection of Nestin ( green ) and Ki67 ( red ) co-labeled cells. D , quantification of HNPC proliferation (Nestin + /Ki67 + ) as mean ± S.E. from three independent experiments. E , HNPCs were transfected as in A in the presence of a Sox2 luciferase reporter construct. Luciferase activity was evaluated 48 h post-transfection as described under “Experimental Procedures.” Results are presented as mean ± S.E. for three independent experiments repeated in triplicate. *, p

    Article Snippet: Primary antibodies against RIT1 (14G7) (Santa Cruz Biotechnology), FLAG, Ki67 (rabbit), BrdU (Sigma), Doublecortin (DCX) (Millipore), Sox2 (Abcam), NeuroD1 (donkey) (Santa Cruz), Nestin (Covance), Akt, phospho-AktS473 , βIII tubulin, ERK1/2, anti-phospho-ERK1/2 (Cell Signaling), and phospho-Sox2T118 (ECM Biosciences) were diluted in blocking serum, incubated overnight with sections at 4 °C, followed by extensive washing with 1× PBS (room temperature).

    Techniques: Expressing, Activity Assay, Mouse Assay, Transfection, Immunohistochemistry, Labeling, Luciferase, Construct

    RIT1 regulates Akt in HNPCs. A , HNPCs were transfected with either EV or FLAG-tagged RIT1 Q97L (CA), and immunohistochemistry was used to detect Nestin ( red ) and active Akt ( green , phospho-Akt (Ser 473 )) (magnification ×20). B , quantification of HNPC proliferation as mean ± S.E. from three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: RIT1 GTPase Regulates Sox2 Transcriptional Activity and Hippocampal Neurogenesis *

    doi: 10.1074/jbc.M116.749770

    Figure Lengend Snippet: RIT1 regulates Akt in HNPCs. A , HNPCs were transfected with either EV or FLAG-tagged RIT1 Q97L (CA), and immunohistochemistry was used to detect Nestin ( red ) and active Akt ( green , phospho-Akt (Ser 473 )) (magnification ×20). B , quantification of HNPC proliferation as mean ± S.E. from three independent experiments. *, p

    Article Snippet: Primary antibodies against RIT1 (14G7) (Santa Cruz Biotechnology), FLAG, Ki67 (rabbit), BrdU (Sigma), Doublecortin (DCX) (Millipore), Sox2 (Abcam), NeuroD1 (donkey) (Santa Cruz), Nestin (Covance), Akt, phospho-AktS473 , βIII tubulin, ERK1/2, anti-phospho-ERK1/2 (Cell Signaling), and phospho-Sox2T118 (ECM Biosciences) were diluted in blocking serum, incubated overnight with sections at 4 °C, followed by extensive washing with 1× PBS (room temperature).

    Techniques: Transfection, Immunohistochemistry

    β8 integrin is required for GSC self-renewal and differentiation in vitro . ( a ) Experimental approach for isolation of β8 high and β8 low primary GBM cells from human samples followed by in vitro culturing and/or intracranial injection. ( b ) Summary of β8 integrin protein expression levels as determined by FACS in 25 different freshly resected primary GBM samples. ( c, d ) β8 high GBM cells from sample HBT14 form spheroids and survive in culture ( c ), whereas β8 low cells do not form spheroids and fail to thrive in culture ( d ). Images shown are of spheroids formed from non-passaged β8 high and β8 low GBM cells. ( e ) Quantitation of β8 integrin-dependent sphere formation in vitro . ( f–i ) Spheroids generated from low-passage β8 high GBM cells (HBT41) were grown in the presence or absence of serum and immunofluorescently labeled with anti-Nestin and anti-GFAP to label neural stem cells and astrocytes ( f, g ) or anti-TUJ1 and anti-neurofilament antibodies to label neurons ( h, i ). ( j ) Neural stem cell markers were quantified by RT-PCR using spheroids generated from low-passage β8 high GBM cells (HBT28) before and after differentiation via serum exposure. ( k ) Serum-induced differentiation of β8 high GBM cells (HBT32) leads to reduced β8 integrin expression at low passages, but β8 integrin expression increases in more differentiated cells at higher passages.

    Journal: Oncogene

    Article Title: Glioblastoma stem cells exploit the αvβ8 integrin-TGFβ1 signaling axis to drive tumor initiation and progression

    doi: 10.1038/onc.2017.248

    Figure Lengend Snippet: β8 integrin is required for GSC self-renewal and differentiation in vitro . ( a ) Experimental approach for isolation of β8 high and β8 low primary GBM cells from human samples followed by in vitro culturing and/or intracranial injection. ( b ) Summary of β8 integrin protein expression levels as determined by FACS in 25 different freshly resected primary GBM samples. ( c, d ) β8 high GBM cells from sample HBT14 form spheroids and survive in culture ( c ), whereas β8 low cells do not form spheroids and fail to thrive in culture ( d ). Images shown are of spheroids formed from non-passaged β8 high and β8 low GBM cells. ( e ) Quantitation of β8 integrin-dependent sphere formation in vitro . ( f–i ) Spheroids generated from low-passage β8 high GBM cells (HBT41) were grown in the presence or absence of serum and immunofluorescently labeled with anti-Nestin and anti-GFAP to label neural stem cells and astrocytes ( f, g ) or anti-TUJ1 and anti-neurofilament antibodies to label neurons ( h, i ). ( j ) Neural stem cell markers were quantified by RT-PCR using spheroids generated from low-passage β8 high GBM cells (HBT28) before and after differentiation via serum exposure. ( k ) Serum-induced differentiation of β8 high GBM cells (HBT32) leads to reduced β8 integrin expression at low passages, but β8 integrin expression increases in more differentiated cells at higher passages.

    Article Snippet: For immunofluorescence, rabbit anti-GFAP (DAKO, Glostrup, Denmark), chicken anti-Nestin (Neuromics, Minneapolis, MN, USA), rat anti-CD31 (BD Biosciences, San Diego, CA, USA), rat anti-CD34 (GeneTex, Irvine, CA, USA), goat anti-Vimentin (R & D Systems), mouse anti-Neuron-specific class III beta-tubulin (TUJ1) from Sigma-Aldrich (Minneapolis, MN, USA) , rabbit anti-Neurofilament (Neuromics), rabbit anti-Laminin (Sigma-Aldrich), mouse anti-NeuN (Chemicon, Billerica, MA, USA), rabbit anti-Myelin Basic Protein (MBP), rabbit anti-Iba1 (Wako, Richmond, VA, USA) were purchased.

    Techniques: In Vitro, Isolation, Injection, Expressing, FACS, Quantitation Assay, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction

    Immunophenotyping of paediatric glioma cell lines. All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H E – haematoxylin and eosin. All images original magnification ×400.

    Journal: PLoS ONE

    Article Title: Molecular and Phenotypic Characterisation of Paediatric Glioma Cell Lines as Models for Preclinical Drug Development

    doi: 10.1371/journal.pone.0005209

    Figure Lengend Snippet: Immunophenotyping of paediatric glioma cell lines. All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H E – haematoxylin and eosin. All images original magnification ×400.

    Article Snippet: The cells underwent a peroxidase block (Dako, Ely, UK) followed by washes in 0.05%Tween in TBS prior to incubation with primary antibody – GFAP (clone 6F2, 1∶50 dilution, Dako); synaptophysin (SY38, 1∶10, Dako); vimentin (V9, 1∶100, Dako), S100 (B32.1, 1∶20, Abcam, Cambridge, UK), nestin (10C2, 1∶400, Miltenyi Biotech, Bergisch Gladbach, Germany), or CD133 (W6B3C1), 1∶40, Miltenyi Biotech).

    Techniques: Staining

    Pax3 negatively regulates p53 protein, but not mRNA levels in ESC just as in mouse embryos. (A) Real time RT-PCR of p53 , Pax3 , and Nestin mRNA in stage 1 (open bars) and stage 3, days 2–8 (solid bars) ESC. Nestin mRNA is expressed in neuroepithelium and in ESC-derived neuronal precursors [29] , [30] and served as a control for a marker of neuroepithelial neuronal precursors. Each mRNA was normalized to rRNA. In (A), (B), and (D) values represent the mean ± SEM (n = 3 culture dishes). *p

    Journal: PLoS ONE

    Article Title: Pax3 Stimulates p53 Ubiquitination and Degradation Independent of Transcription

    doi: 10.1371/journal.pone.0029379

    Figure Lengend Snippet: Pax3 negatively regulates p53 protein, but not mRNA levels in ESC just as in mouse embryos. (A) Real time RT-PCR of p53 , Pax3 , and Nestin mRNA in stage 1 (open bars) and stage 3, days 2–8 (solid bars) ESC. Nestin mRNA is expressed in neuroepithelium and in ESC-derived neuronal precursors [29] , [30] and served as a control for a marker of neuroepithelial neuronal precursors. Each mRNA was normalized to rRNA. In (A), (B), and (D) values represent the mean ± SEM (n = 3 culture dishes). *p

    Article Snippet: Primers and probes for rRNA and Nestin were obtained from PerkinElmer.

    Techniques: Quantitative RT-PCR, Derivative Assay, Marker