anti-nestin Search Results


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  • 99
    Millipore anti nestin mouse ab
    Distribution and localization of <t>DOCK7</t> in the developing mouse cortex. ( a ) DOCK7 levels in cortical lysates at different developmental stages. γ-tubulin was used as a loading control. Full-length Western blot is shown in Supplementary Figure 12 . ( b ) Coronal cryosections of the neocortex at E13.5 immunostained for DOCK7 and neural stem/progenitor marker <t>nestin</t> or neuronal marker class III β-tubulin Tuj1. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. ( c ) Coronal brain slices of the neocortex at E13.5 immunostained for DOCK7 and centrosomal marker γ-tubulin, and counterstained with DAPI. An enlarged view of the apical endfeet of VZ progenitors is shown. ( d ) Cultured cortical progenitors isolated from E13.5 neocortices immunostained for DOCK7 and centrosomal marker pericentrin, and counterstained with DAPI. Scale bars: 15 µm in b; 10 µm in c; 5 µm in d.
    Anti Nestin Mouse Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam anti nestin antibody
    CAR expression in the sub-ventricular zone and the rostral migratory stream. (A–D) Colocalization of CAR (green), <t>DCX</t> (red), and Sox2 (blue) in the SVZ. Both CAR and DCX are present in the same cells (white arrows) that also express low levels of Sox2 (Z-stack 10 μm). (E–H) Colocalization of CAR (green) and <t>nestin</t> (red) in the SVZ. Nestin and CAR colocalization in some cells but not in all. And both CAR and nestin colocalize in those cells with lower levels of Sox2 (blue) (Z-stack 10 μm). (I–L) Colocalization of CAR (green) and GFAP (red) in the SVZ. Also, there was no presence of NeuN (blue) in the CAR + cells (Z-stack 10 μm). (M–P) Expression of CAR (green), GFAP (red), and NeuN (blue) in the RMS. On the contrary that we observed in the SVZ, in the RMS there was a lack of CAR expression in the GFAP-ir fibers. As in the SVZ, CAR-ir cells were not NeuN immunoreactive (Z-stack 10 μm). Calibration bars: (A–P) : 10 μm.
    Anti Nestin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend anti nestin
    Relapsing-remitting multiple sclerosis (RR-MS) CD49d + CD154 + lymphocytes affect maturing oligodendrocyte precursor cells (OPCs), resulting in dysregulation of myelin production by mature oligodendrocytes (OLs). ( A ) RR-MS CD49d + CD154 + , contrary to healthy control (HC) lymphocytes, affected myelin basic protein (MBP) and proteolipid protein (PLP) but not myelin oligodendrocyte glycoprotein (MOG) synthesis by human OPCs (hOPCs). ( B ) MBP/PLP index more clearly exhibited disproportion in myelin protein synthesis. ( C ) Immunocytochemical (ICC) analysis of <t>nestin,</t> <t>O4</t> and GFAP expression in hOPCs demonstrated that RR-MS CD49d + CD154 + lymphocytes did not affect hOPC maturation, but caused dysregulation of MBP and PLP synthesis by mature OLs. All data are presented as means ± SD.
    Anti Nestin, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antinestin
    Relapsing-remitting multiple sclerosis (RR-MS) CD49d + CD154 + lymphocytes affect maturing oligodendrocyte precursor cells (OPCs), resulting in dysregulation of myelin production by mature oligodendrocytes (OLs). ( A ) RR-MS CD49d + CD154 + , contrary to healthy control (HC) lymphocytes, affected myelin basic protein (MBP) and proteolipid protein (PLP) but not myelin oligodendrocyte glycoprotein (MOG) synthesis by human OPCs (hOPCs). ( B ) MBP/PLP index more clearly exhibited disproportion in myelin protein synthesis. ( C ) Immunocytochemical (ICC) analysis of <t>nestin,</t> <t>O4</t> and GFAP expression in hOPCs demonstrated that RR-MS CD49d + CD154 + lymphocytes did not affect hOPC maturation, but caused dysregulation of MBP and PLP synthesis by mature OLs. All data are presented as means ± SD.
    Antinestin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti nestin
    p38 protein is expressed in doublecortin (Dcx)-positive NPCs in the adult brain. Brain slices were prepared from adult mice, and immunohistochemical analysis was performed with anti-p38, anti-Dcx, <t>anti-GFAP,</t> and <t>anti-nestin</t> antibodies. p38 was expressed specifically in Dcx-positive NPCs of the subventricular zone ( a–c ) and RMS ( d ) rather than in GFAP- ( e–h ) or nestin-positive ( i–k ) neural stem cells. ( l ) Negative control staining using only secondary antibody. CC, corpus callosum; LV, lateral ventricle; ST, striatum; SVZ, subventricular zone. Scale bar = 20 μm ( b–k ), 100 μm ( a,e,i,l ).
    Anti Nestin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti nestin
    Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or <t>Map2</t> (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and <t>Nestin</t> (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P
    Anti Nestin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank anti nestin
    Abnormal development of neural stem cell-derived neurospheres from Raldh2 −/− spinal cords. Neurospheres were derived from dorsal cervical/brachial spinal cord from E12.5 WT and Raldh2 −/− embryos. (A,B) After growth of dissociated progenitor cells in suspension, in 6-well plates, for 10 days in a defined selective medium (see Materials and Methods ), the number of spheres developing from Raldh2 −/− embryos spinal cords is decreased by 25% compared to WT (E: 11.1±0.88 spheres/well for WT; 8.40±1.50 for mutants; n = 9; T- test: P value = 0,0009). (C,D) To further study progenitor cell differentiation, spheres were plated onto laminin-coated coverslips for 3 days, after 10 days of growth in suspension. Raldh2 −/− derived spheres exhibited reduced number of cells compared to WT (F: one quadrant of a plated WT sphere is composed of 105.4±16.6 cells, against 40.9±6,82 cells in a Raldh2 −/− sphere; DAPI positive nuclei were counted using ImageJ software; n = 9; P = 1.6×10 −5 ). (G–O) After growth onto laminin coated coverslips for 3 days, cells were processed for immunocytochemistry. <t>Nestin+</t> cells (I,M) are increased by 20% in the Raldh2 −/− derived spheres, while <t>TuJ1+</t> cells (J,N) are decreased by half. (H,L, DAPI staining; K,O, merged images). (G) After counting and normalization for total cell numbers, assessed by DAPI positive cell nuclei, one quadrant of a WT sphere contains 41.6±14 Nestin+ and 42.0±9.76 TuJ1+ cells, against 57.9±9.21 and 25.9±6,66 in a Raldh2 −/− sphere (n = 9; P = 0,001 and 0,007, respectively).
    Anti Nestin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen anti nestin
    Mitotic neuroblasts in the embryonic mouse cortex with lagging chromosomes. ( A and B ) Low magnification (×20) micrographs of the embryonic cerebral cortex. Immunofluorescence for the intermediate filament protein <t>nestin</t> ( A ), a neural progenitor cell marker, illustrates the distribution of neuroblasts in the ventricular zone (VZ) of the embryonic cerebral cortex. <t>Phospho-H3</t> labeling ( B , red) reveals mitotic neuroblasts concentrated at the ventricular surface (bottom) of the VZ. Nuclei are counterstained with DAPI (blue). ( C – F ) High magnification (×100) Z stacks from deconvolution microscopy of phospho-H3-labeled mitotic figures at the bottom of the VZ reveal morphologically normal prometaphase/metaphase ( C ) and anaphase ( D ) profiles. In addition, lagging chromosomes (arrows) are readily observed in prometaphase/metaphase ( E ) and anaphase ( F ) profiles.
    Anti Nestin, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti nestin
    <t>PATZ1</t> localization in the stem cell compartment of GBM Representative confocal microscopy images of GBM (two samples with high PATZ1 score (+++) and GBM-associated normal tissues stained with <t>anti-NESTIN</t> (A, E) , anti-OCT-4 (I) and anti-PATZ1 (B, F, J) . (D, H, L) Merge images showing co-localization of PATZ1 with either NESTIN or OCT-4. Scale bar: 20 μm. (C, G, K) Nuclei are depicted by the blue fluorescence of DAPI.
    Anti Nestin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 524 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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    92
    Merck KGaA nestin
    <t>Nestin</t> and <t>CD15</t> expression after in vitro treatment with TMZ, Dox and a combination of both drugs. Upper panels show western blot analysis of GBM non-CSCs under different treatment conditions (50μM TMZ, 50μM Dox or 50μM each), Tubulin represents the loading control. Lower panels are results from densitometric scanning analyses of the western blot signals. Results are given as relative expression to untreated control cells.
    Nestin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    OriGene nestin
    Immunohistochemical staining of neuron-like cells in treatment groups for <t>NSE,</t> <t>nestin,</t> and GFAP, respectively. The arrows in figures indicate neuron-like cells.
    Nestin, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti nestin
    Characterization of spontaneous mitochondrial SO flashes in NPCs. (A): Dissociated NPCs at day 3 in culture were immunostained with antibodies against <t>Sox2,</t> <t>nestin</t> and BrdU (after a 16 h exposure to 10 μM BrdU) (green), which are markers of proliferating
    Anti Nestin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Distribution and localization of DOCK7 in the developing mouse cortex. ( a ) DOCK7 levels in cortical lysates at different developmental stages. γ-tubulin was used as a loading control. Full-length Western blot is shown in Supplementary Figure 12 . ( b ) Coronal cryosections of the neocortex at E13.5 immunostained for DOCK7 and neural stem/progenitor marker nestin or neuronal marker class III β-tubulin Tuj1. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. ( c ) Coronal brain slices of the neocortex at E13.5 immunostained for DOCK7 and centrosomal marker γ-tubulin, and counterstained with DAPI. An enlarged view of the apical endfeet of VZ progenitors is shown. ( d ) Cultured cortical progenitors isolated from E13.5 neocortices immunostained for DOCK7 and centrosomal marker pericentrin, and counterstained with DAPI. Scale bars: 15 µm in b; 10 µm in c; 5 µm in d.

    Journal: Nature neuroscience

    Article Title: DOCK7 interacts with TACC3 to regulate interkinetic nuclear migration and cortical neurogenesis

    doi: 10.1038/nn.3171

    Figure Lengend Snippet: Distribution and localization of DOCK7 in the developing mouse cortex. ( a ) DOCK7 levels in cortical lysates at different developmental stages. γ-tubulin was used as a loading control. Full-length Western blot is shown in Supplementary Figure 12 . ( b ) Coronal cryosections of the neocortex at E13.5 immunostained for DOCK7 and neural stem/progenitor marker nestin or neuronal marker class III β-tubulin Tuj1. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. ( c ) Coronal brain slices of the neocortex at E13.5 immunostained for DOCK7 and centrosomal marker γ-tubulin, and counterstained with DAPI. An enlarged view of the apical endfeet of VZ progenitors is shown. ( d ) Cultured cortical progenitors isolated from E13.5 neocortices immunostained for DOCK7 and centrosomal marker pericentrin, and counterstained with DAPI. Scale bars: 15 µm in b; 10 µm in c; 5 µm in d.

    Article Snippet: We used the following primary antibodies: anti-DOCK7 (rabbit, 1:500); anti-nestin (mouse, 1:200, Chemicon); anti-Tuj1 (mouse 1:1,500, Covance); anti-γ-tubulin (mouse, 1:2,000, Sigma); anti-GFP (chicken, 1:500, Aves Labs); anti-BrdU (rat, 1:500, AbD Serotec); anti-PH3 (rabbit, 1:1,000, Millipore); anti-Ki67 (rabbit, 1:250, Vector Laboratories); anti-Pax6 (rabbit, 1:500, Covance); anti-Tbr2 (rabbit, 1:300, Abcam); anti-RFP (rabbit, 1:500, Rockland); anti-pericentrin (mouse, 1:1,000, BD Biosciences); anti-TACC3 (mouse, 1:300, Santa Cruz); anti-α-tubulin (mouse, 1:2,000, Sigma); anti-ZO-1 (mouse, 1:100, Invitrogen); anti-GFAP (chicken, 1:500, Aves Labs).

    Techniques: Western Blot, Marker, Cell Culture, Isolation

    Double immunofluorescence staining for Luc/Nestin and Luc/Osteocalcin (DAPI for nucleuses). a In the Sys group, Luc + /Nestin + cells (the red-yellow cells pointed out by red arrows ), the systemically injected allogenic Luc-MSCs, had migrated to the fracture site. b In the Loc group, Luc + /Nestin + cells ( red arrows ), the injected Luc-MSCs through local sites, remained at 5 weeks following the fracture. Luc + /Nestin − cells (the green cells pointed out by white arrows in a, b) were observed, which meant that some injected Luc-MSCs had differentiated to other types of cells in both MSC injection groups. c , d Luciferase-positive cells were not observed in the PBS group ( c ) and the negative control ( d ), but Luc − /Nestin + cells ( white arrows in c), autogenetic MSCs, existed in the callus in the PBS control group. e , f Luc + /Osteocalcin + cells (the red-yellow cells pointed out by red arrows ) were observed both in the Sys group ( e ) and the Loc group ( f ), which meant that some injected Luc-MSCs had differentiated to osteoblasts. Luc + /Osteocalcin − cells, the green cells ( white arrows ), were also found in MSC injection groups, which meant that some injected Luc cells remained at 5 weeks following the fracture. g , h Luciferase-positive cells were not observed in the PBS control group ( g ) and the negative control ( h ), but Luc − /Osteocalcin + cells, autogenetic osteoblasts, were found in the callus. Scale bar: 20 μm. DAPI 4′,6-diamidino-2-phenylindole, Luc-MSC Luciferase labeled mesenchymal stem cell, MSC mesenchymal stem cell, PBS phosphate-buffered saline

    Journal: Stem Cell Research & Therapy

    Article Title: The fate of systemically administrated allogeneic mesenchymal stem cells in mouse femoral fracture healing

    doi: 10.1186/s13287-015-0198-7

    Figure Lengend Snippet: Double immunofluorescence staining for Luc/Nestin and Luc/Osteocalcin (DAPI for nucleuses). a In the Sys group, Luc + /Nestin + cells (the red-yellow cells pointed out by red arrows ), the systemically injected allogenic Luc-MSCs, had migrated to the fracture site. b In the Loc group, Luc + /Nestin + cells ( red arrows ), the injected Luc-MSCs through local sites, remained at 5 weeks following the fracture. Luc + /Nestin − cells (the green cells pointed out by white arrows in a, b) were observed, which meant that some injected Luc-MSCs had differentiated to other types of cells in both MSC injection groups. c , d Luciferase-positive cells were not observed in the PBS group ( c ) and the negative control ( d ), but Luc − /Nestin + cells ( white arrows in c), autogenetic MSCs, existed in the callus in the PBS control group. e , f Luc + /Osteocalcin + cells (the red-yellow cells pointed out by red arrows ) were observed both in the Sys group ( e ) and the Loc group ( f ), which meant that some injected Luc-MSCs had differentiated to osteoblasts. Luc + /Osteocalcin − cells, the green cells ( white arrows ), were also found in MSC injection groups, which meant that some injected Luc cells remained at 5 weeks following the fracture. g , h Luciferase-positive cells were not observed in the PBS control group ( g ) and the negative control ( h ), but Luc − /Osteocalcin + cells, autogenetic osteoblasts, were found in the callus. Scale bar: 20 μm. DAPI 4′,6-diamidino-2-phenylindole, Luc-MSC Luciferase labeled mesenchymal stem cell, MSC mesenchymal stem cell, PBS phosphate-buffered saline

    Article Snippet: In brief, slides were blocked by 5 % donkey serum in 1 % BSA for 20 minutes, incubated with the goat anti-Luciferase antibody (1:300; Santa Cruz Biotechnology, Inc.) and the rabbit anti-Osteocalcin antibody (1:300; Santa Cruz Biotechnology, Inc.) or the rabbit anti-Nestin antibody (1:300; Sigma-Aldrich) overnight at 4 °C, washed with PBS three times, and incubated with the donkey anti-goat IgG-FITC (1:1000; Santa Cruz Biotechnology, Inc.) and Cy3 donkey anti-rabbit IgG (1:1000; Life Technologies, Carlsbad, CA, USA) for 60 minutes at room temperature in the dark.

    Techniques: Double Immunofluorescence Staining, Injection, Luciferase, Negative Control, Labeling

    Generation and differentiation of NKX2.5-GFP iPS cells (A) iPS cell colony 8 days after viral infection of adult skin fibroblasts. (B) SSEA-1 or (C) Oct-4 immunocytochemistry. (D) Endogenous expression of pluripotency genes by qPCR in four iPS cell lines relative to ES cells. HP= high passage. (E) FACS plot of negative control (iPS cells stained with 2 nd antibody only). (F) FACS plot of SSEA-1 + iPS cells. (G–L) H E staining of teratoma sections: (G) low magnification view of Nkx2.5-GFP iPS-derived teratoma; (H) neural rosettes; (I) adipose tissue; (J) cartilage; (K) muscle (and tubular epithelium); (L) tubular epithelium. (M–Q) Immunostaining of teratoma sections for (M) nestin (neural progenitors); (N) GFAP (glia); (O) smooth muscle actin (SMA); (P) α-actinin (red), GFP (green); (Q) alpha fetal-protein (AFP, endoderm). (R) Percent beating EBs with different induction methods: hanging drop (HD), suspension (SUSP), or END-2 co-culture (END). (S–W) Derivatives of all three germ layers in EBs in vitro demonstrated by immunostaining with indicated markers. (S–U) Suspension EBs (V–W) on END-2 cells immunostained with indicated markers.

    Journal: Circulation research

    Article Title: Reporter-Based Isolation of Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Cardiac Progenitors Reveals Limited Gene Expression Variance

    doi: 10.1161/CIRCRESAHA.109.215434

    Figure Lengend Snippet: Generation and differentiation of NKX2.5-GFP iPS cells (A) iPS cell colony 8 days after viral infection of adult skin fibroblasts. (B) SSEA-1 or (C) Oct-4 immunocytochemistry. (D) Endogenous expression of pluripotency genes by qPCR in four iPS cell lines relative to ES cells. HP= high passage. (E) FACS plot of negative control (iPS cells stained with 2 nd antibody only). (F) FACS plot of SSEA-1 + iPS cells. (G–L) H E staining of teratoma sections: (G) low magnification view of Nkx2.5-GFP iPS-derived teratoma; (H) neural rosettes; (I) adipose tissue; (J) cartilage; (K) muscle (and tubular epithelium); (L) tubular epithelium. (M–Q) Immunostaining of teratoma sections for (M) nestin (neural progenitors); (N) GFAP (glia); (O) smooth muscle actin (SMA); (P) α-actinin (red), GFP (green); (Q) alpha fetal-protein (AFP, endoderm). (R) Percent beating EBs with different induction methods: hanging drop (HD), suspension (SUSP), or END-2 co-culture (END). (S–W) Derivatives of all three germ layers in EBs in vitro demonstrated by immunostaining with indicated markers. (S–U) Suspension EBs (V–W) on END-2 cells immunostained with indicated markers.

    Article Snippet: Immunocytochemistry was performed as described with cells on coverslips or cryosections of EBs (n≥3 biological samples per cell line) using the following antibodies: Oct-4 (mouse, 1:100, Santa Cruz), SSEA-1 (mouse IgM, 1:50, Chemicon), α-actinin (mouse, 1:400, Sigma), SMA (mouse, undiluted, DAKO), Nestin (mouse, 1:100, Chemicon), GFAP (rabbit, 1:100, DAKO), AFP (mouse, 1:100, R & D), GFP (rabbit, 1:500, alexa-488 conjugated, Invitrogen).

    Techniques: Infection, Immunocytochemistry, Expressing, Real-time Polymerase Chain Reaction, FACS, Negative Control, Staining, Derivative Assay, Immunostaining, Co-Culture Assay, In Vitro

    CAR expression in the sub-ventricular zone and the rostral migratory stream. (A–D) Colocalization of CAR (green), DCX (red), and Sox2 (blue) in the SVZ. Both CAR and DCX are present in the same cells (white arrows) that also express low levels of Sox2 (Z-stack 10 μm). (E–H) Colocalization of CAR (green) and nestin (red) in the SVZ. Nestin and CAR colocalization in some cells but not in all. And both CAR and nestin colocalize in those cells with lower levels of Sox2 (blue) (Z-stack 10 μm). (I–L) Colocalization of CAR (green) and GFAP (red) in the SVZ. Also, there was no presence of NeuN (blue) in the CAR + cells (Z-stack 10 μm). (M–P) Expression of CAR (green), GFAP (red), and NeuN (blue) in the RMS. On the contrary that we observed in the SVZ, in the RMS there was a lack of CAR expression in the GFAP-ir fibers. As in the SVZ, CAR-ir cells were not NeuN immunoreactive (Z-stack 10 μm). Calibration bars: (A–P) : 10 μm.

    Journal: Frontiers in Neuroanatomy

    Article Title: Location of the Cell Adhesion Molecule “Coxsackievirus and Adenovirus Receptor” in the Adult Mouse Brain

    doi: 10.3389/fnana.2020.00028

    Figure Lengend Snippet: CAR expression in the sub-ventricular zone and the rostral migratory stream. (A–D) Colocalization of CAR (green), DCX (red), and Sox2 (blue) in the SVZ. Both CAR and DCX are present in the same cells (white arrows) that also express low levels of Sox2 (Z-stack 10 μm). (E–H) Colocalization of CAR (green) and nestin (red) in the SVZ. Nestin and CAR colocalization in some cells but not in all. And both CAR and nestin colocalize in those cells with lower levels of Sox2 (blue) (Z-stack 10 μm). (I–L) Colocalization of CAR (green) and GFAP (red) in the SVZ. Also, there was no presence of NeuN (blue) in the CAR + cells (Z-stack 10 μm). (M–P) Expression of CAR (green), GFAP (red), and NeuN (blue) in the RMS. On the contrary that we observed in the SVZ, in the RMS there was a lack of CAR expression in the GFAP-ir fibers. As in the SVZ, CAR-ir cells were not NeuN immunoreactive (Z-stack 10 μm). Calibration bars: (A–P) : 10 μm.

    Article Snippet: The following primary antibodies were used for immunofluorescence: (1) a goat anti-CXADR (CAR) (1:100, R & D systems, AF2654, RRID:AB_2245567 , Lot VFT0119071); (2) a rabbit anti-glial fibrillary acidic protein (GFAP) (1:1000, DAKO, Z0334, RRID:AB_10013382 ); (3) a mouse anti-SOX2 (1:200, ABCAM, ab171380, RRID:AB_2732072 ); (4) a mouse anti-NeuN (1:500, ABCAM, ab104224, RRID:AB_10711040 ); (5) a rabbit anti-doublecortin (DCX) (1:500, ABCAM, ab18723, RRID:AB_732011 ); and (6) a chicken anti-nestin (1:1000, ABCAM, ab134017, RRID:AB_2753197).

    Techniques: Expressing

    CAR expression in the subgranular layer of the dentate gyrus. (A–D) Expression of CAR (green), GFAP (red), and NeuN (blue) in the SGZ of the dentate gyrus. There are some fibers GFAP + that also are CAR + (white arrows). Some CAR + cells are also expressing Neun (yellow arrows). (E–H) Colocalization of CAR (green), nestin (red), and DAPI (white) in the SGZ. Nestin and CAR colocalization in some cells (yellow arrows). (I–L) Colocalization of CAR (green), DCX (red), and DAPI (white) in the SGZ. Both CAR and DCX are present in the same cells (white arrows). Calibration bars: (A–L) : 10 μm.

    Journal: Frontiers in Neuroanatomy

    Article Title: Location of the Cell Adhesion Molecule “Coxsackievirus and Adenovirus Receptor” in the Adult Mouse Brain

    doi: 10.3389/fnana.2020.00028

    Figure Lengend Snippet: CAR expression in the subgranular layer of the dentate gyrus. (A–D) Expression of CAR (green), GFAP (red), and NeuN (blue) in the SGZ of the dentate gyrus. There are some fibers GFAP + that also are CAR + (white arrows). Some CAR + cells are also expressing Neun (yellow arrows). (E–H) Colocalization of CAR (green), nestin (red), and DAPI (white) in the SGZ. Nestin and CAR colocalization in some cells (yellow arrows). (I–L) Colocalization of CAR (green), DCX (red), and DAPI (white) in the SGZ. Both CAR and DCX are present in the same cells (white arrows). Calibration bars: (A–L) : 10 μm.

    Article Snippet: The following primary antibodies were used for immunofluorescence: (1) a goat anti-CXADR (CAR) (1:100, R & D systems, AF2654, RRID:AB_2245567 , Lot VFT0119071); (2) a rabbit anti-glial fibrillary acidic protein (GFAP) (1:1000, DAKO, Z0334, RRID:AB_10013382 ); (3) a mouse anti-SOX2 (1:200, ABCAM, ab171380, RRID:AB_2732072 ); (4) a mouse anti-NeuN (1:500, ABCAM, ab104224, RRID:AB_10711040 ); (5) a rabbit anti-doublecortin (DCX) (1:500, ABCAM, ab18723, RRID:AB_732011 ); and (6) a chicken anti-nestin (1:1000, ABCAM, ab134017, RRID:AB_2753197).

    Techniques: Expressing

    Relapsing-remitting multiple sclerosis (RR-MS) CD49d + CD154 + lymphocytes affect maturing oligodendrocyte precursor cells (OPCs), resulting in dysregulation of myelin production by mature oligodendrocytes (OLs). ( A ) RR-MS CD49d + CD154 + , contrary to healthy control (HC) lymphocytes, affected myelin basic protein (MBP) and proteolipid protein (PLP) but not myelin oligodendrocyte glycoprotein (MOG) synthesis by human OPCs (hOPCs). ( B ) MBP/PLP index more clearly exhibited disproportion in myelin protein synthesis. ( C ) Immunocytochemical (ICC) analysis of nestin, O4 and GFAP expression in hOPCs demonstrated that RR-MS CD49d + CD154 + lymphocytes did not affect hOPC maturation, but caused dysregulation of MBP and PLP synthesis by mature OLs. All data are presented as means ± SD.

    Journal: Cells

    Article Title: MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration

    doi: 10.3390/cells8121508

    Figure Lengend Snippet: Relapsing-remitting multiple sclerosis (RR-MS) CD49d + CD154 + lymphocytes affect maturing oligodendrocyte precursor cells (OPCs), resulting in dysregulation of myelin production by mature oligodendrocytes (OLs). ( A ) RR-MS CD49d + CD154 + , contrary to healthy control (HC) lymphocytes, affected myelin basic protein (MBP) and proteolipid protein (PLP) but not myelin oligodendrocyte glycoprotein (MOG) synthesis by human OPCs (hOPCs). ( B ) MBP/PLP index more clearly exhibited disproportion in myelin protein synthesis. ( C ) Immunocytochemical (ICC) analysis of nestin, O4 and GFAP expression in hOPCs demonstrated that RR-MS CD49d + CD154 + lymphocytes did not affect hOPC maturation, but caused dysregulation of MBP and PLP synthesis by mature OLs. All data are presented as means ± SD.

    Article Snippet: Anti-MOG (mouse, Santa Cruz Biotechnology, Dallas, TX, USA), anti-O4 (clone O4, R & D Systems), anti-Nestin (rabbit, BioLegend, San Diego, CA, USA), anti-Dicer1 (clone F10, Santa Cruz Biotechnology, Dallas, TX, USA), phalloidin (F-actin)/TR (Invitrogen), anti-Ago2 (rabbit, Abcam, Cambridge, UK), anti-GFAP (clone H50, Santa Cruz Biotechnology, Dallas, TX, USA), and rat IgG2b (Invitrogen, Carlsbad, CA, USA) as negative isotype control, were used.

    Techniques: Mass Spectrometry, Plasmid Purification, Immunocytochemistry, Expressing

    Mouse brain-infiltrating mononuclear cells (BMCs) affect myelin protein synthesis by mouse OPCs (mOPCs). ( A ) BMCs from Experimental Autoimmune Encephalomyelitis (EAE) mice in the third week after the peak of the disease (remyelination phase) were cocultured with OPCs isolated from the healthy mouse newborns (mOPCs). (A1, left panel) Phenotypic analysis of BMCs (region R1) divided into B cells (B220, region R2) and T cells (TCR-β, region R3). (A1, right panel) CD3 + and CD19 + cell rate analysis in isolated BMCs. (A2) Memory B cells (mCD19 + ) were positively selected, and unlabeled cells additionally sorted by negative selection into CD3 + lymphocytes from BMCs. ( B , upper panel) In contrast to immature cells, mature OLs exhibited MBP/PLP/MOG expression (markers of the late stage of differentiation), O4 expression (marker of mature cells), and negative signal for nestin (marker of OL precursor cells) and GFAP (astrocyte-specific marker). ( B , low panel) EAE CD3 + /mCD19 + and CD3 + cells alone down- PLP and upregulated MBP expression, but did not affect O4 or nestin. Maturation process was associated with formation of actin-like microfilaments (labelled with phalloidin). Confocal z-stack analysis confirmed that EAE CD3 + /mCD19 + BMC-derived cells did not affect O4 accumulation in the intracellular space and microfilament formation during OPC maturation. ( C ) mRNA myelin protein analysis proved that EAE CD3 + /mCD19 + and CD3 + BMC-derived cells up- MBP and downregulated PLP, but had no influence on MOG synthesis. All data are presented as means ± SD.

    Journal: Cells

    Article Title: MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration

    doi: 10.3390/cells8121508

    Figure Lengend Snippet: Mouse brain-infiltrating mononuclear cells (BMCs) affect myelin protein synthesis by mouse OPCs (mOPCs). ( A ) BMCs from Experimental Autoimmune Encephalomyelitis (EAE) mice in the third week after the peak of the disease (remyelination phase) were cocultured with OPCs isolated from the healthy mouse newborns (mOPCs). (A1, left panel) Phenotypic analysis of BMCs (region R1) divided into B cells (B220, region R2) and T cells (TCR-β, region R3). (A1, right panel) CD3 + and CD19 + cell rate analysis in isolated BMCs. (A2) Memory B cells (mCD19 + ) were positively selected, and unlabeled cells additionally sorted by negative selection into CD3 + lymphocytes from BMCs. ( B , upper panel) In contrast to immature cells, mature OLs exhibited MBP/PLP/MOG expression (markers of the late stage of differentiation), O4 expression (marker of mature cells), and negative signal for nestin (marker of OL precursor cells) and GFAP (astrocyte-specific marker). ( B , low panel) EAE CD3 + /mCD19 + and CD3 + cells alone down- PLP and upregulated MBP expression, but did not affect O4 or nestin. Maturation process was associated with formation of actin-like microfilaments (labelled with phalloidin). Confocal z-stack analysis confirmed that EAE CD3 + /mCD19 + BMC-derived cells did not affect O4 accumulation in the intracellular space and microfilament formation during OPC maturation. ( C ) mRNA myelin protein analysis proved that EAE CD3 + /mCD19 + and CD3 + BMC-derived cells up- MBP and downregulated PLP, but had no influence on MOG synthesis. All data are presented as means ± SD.

    Article Snippet: Anti-MOG (mouse, Santa Cruz Biotechnology, Dallas, TX, USA), anti-O4 (clone O4, R & D Systems), anti-Nestin (rabbit, BioLegend, San Diego, CA, USA), anti-Dicer1 (clone F10, Santa Cruz Biotechnology, Dallas, TX, USA), phalloidin (F-actin)/TR (Invitrogen), anti-Ago2 (rabbit, Abcam, Cambridge, UK), anti-GFAP (clone H50, Santa Cruz Biotechnology, Dallas, TX, USA), and rat IgG2b (Invitrogen, Carlsbad, CA, USA) as negative isotype control, were used.

    Techniques: Mouse Assay, Isolation, Selection, Plasmid Purification, Expressing, Marker, Derivative Assay

    p38 protein is expressed in doublecortin (Dcx)-positive NPCs in the adult brain. Brain slices were prepared from adult mice, and immunohistochemical analysis was performed with anti-p38, anti-Dcx, anti-GFAP, and anti-nestin antibodies. p38 was expressed specifically in Dcx-positive NPCs of the subventricular zone ( a–c ) and RMS ( d ) rather than in GFAP- ( e–h ) or nestin-positive ( i–k ) neural stem cells. ( l ) Negative control staining using only secondary antibody. CC, corpus callosum; LV, lateral ventricle; ST, striatum; SVZ, subventricular zone. Scale bar = 20 μm ( b–k ), 100 μm ( a,e,i,l ).

    Journal: Scientific Reports

    Article Title: Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells

    doi: 10.1038/srep24279

    Figure Lengend Snippet: p38 protein is expressed in doublecortin (Dcx)-positive NPCs in the adult brain. Brain slices were prepared from adult mice, and immunohistochemical analysis was performed with anti-p38, anti-Dcx, anti-GFAP, and anti-nestin antibodies. p38 was expressed specifically in Dcx-positive NPCs of the subventricular zone ( a–c ) and RMS ( d ) rather than in GFAP- ( e–h ) or nestin-positive ( i–k ) neural stem cells. ( l ) Negative control staining using only secondary antibody. CC, corpus callosum; LV, lateral ventricle; ST, striatum; SVZ, subventricular zone. Scale bar = 20 μm ( b–k ), 100 μm ( a,e,i,l ).

    Article Snippet: Sections were then incubated for 24 hr at 4 °C with anti-p38α (sc-535, rabbit polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA), anti-doublecortin (sc-8066, goat polyclonal; Santa Cruz Biotechnology), anti-GFAP (glial fibrillary acidic protein, G3893, mouse monoclonal IgG; Sigma-Aldrich), and anti-nestin (2Q178, mouse monoclonal IgG; Abcam, Cambridge, UK) antibodies.

    Techniques: Mouse Assay, Immunohistochemistry, Negative Control, Staining

    Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P

    Journal: Nature Communications

    Article Title: Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

    doi: 10.1038/s41467-017-02720-9

    Figure Lengend Snippet: Klf4 down-regulation enhances neurogenesis in vivo and in vitro. a (Upper) Fixed coronal sections from E11.5 (left) or E14.5 (right) mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), or Map2 (green). Nuclear staining is shown by DAPI (blue). (Lower) Quantification of data shown above (E11.5: Klf4 fl/+ ( WT ), n = 4–8, Klf4 conditional knockout ( cKO ), n = 3–8, and E14.5: Klf4 fl/+ ( WT ), n = 3–9, Klf4 cKO , n = 4–6). Scale bars, 50 μm. b (Left) Analysis of Pax6 in sections described in a . Scale bar, 50 μm. (Right) Quantification of data shown on the left (E11.5: WT , n = 5, Klf4 cKO , n = 5, and E14.5: WT , n = 5, Klf4 cKO , n = 3). c (Upper) Immunostaining with Tuj1 or Map2 (both red) antibodies in WT and Klf4 cKO NPCs in undifferentiation and differentiation conditions. Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of Tuj1-positive ( WT -Un, n = 4, WT -D2, n = 4, WT -D4, n = 6, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 3, Klf4 cKO -D4, n = 8) and Map2-positive ( WT -Un, n = 5, WT -D2, n = 5, WT -D4, n = 3, Klf4 cKO -Un, n = 4, Klf4 cKO -D2, n = 4, Klf4 cKO -D4, n = 5) cells in panels above. d Immunostaining with Ki67 (red) and Nestin (green) antibodies in WT and Klf4 cKO NPCs. DAPI (blue). Scale bars, 25 μm. (Right) Quantification of Ki67/Nestin double-positive cells in analysis shown at left ( n = 4). e Single WT and Klf4 cKO NPCs were separated by serial dilution and neurosphere formation was induced for 7 days in vitro (DIV). Relative size of primary spheres grown to 7 DIV was quantified by Image J Software. Scale bars, blue (100 pixel), red (200 pixel). f qPCR analysis of indicated mRNAs in WT and Klf4 cKO NPCs. Values correspond to mean ± SD. ANOVA tests were performed to calculate significance (* P

    Article Snippet: Antibodies and reagents Antibodies used in this study were anti-Klf4 (rabbit polyclonal 1:500; Gene Tex, Irvine, CA, USA), anti-Flag (mouse, 1:1000; Sigma, St. Louis, MO, USA), anti-HA (rabbit, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Stau1 (rabbit, 1:500; Thermo Scientific, Rockford, IL, USA), anti-Map2 (rabbit polyclonal 1:200, Chemicon, Temecula, CA, USA), anti-Nestin (mouse monoclonal 1:350, BD Biosciences, San Jose, CA, USA), anti-Tuj1 (mouse polyclonal 1:200; Covance, Princeton, NJ, USA), anti-Tbr1 (rabbit polyclonal 1:200; Abcam Ltd, Cambridge, MA, USA), anti-Pax6 (rabbit polyclonal 1:200; Abcam Ltd), anti-Ddx5 (rabbit polyclonal 1:1000; Abcam Ltd), anti-Ddx17 (rabbit polyclonal 1:1000; Abcam Ltd), anti-GST (rabbit polyclonal 1:500; Santa Cruz Biotechnology), anti-BrdU (mouse monoclonal 1:500; Abcam Ltd), anti-Dlx1 (mouse monoclonal 1:250; Abnova, Walnut, CA, USA), anti-Dlx2 (rabbit polyclonal, 1:250; Abcam Ltd), anti-Ctip2 (Rat 1:200; Abcam Ltd), anti-cleaved caspase-3 (mouse 1:200; R & D Systems, Inc., Minneapolis, MN, USA), anti-UPF1 (rabbit 1:200; Cell Signaling Technology Inc., Danvers, MA, USA), and anti-α-tubulin (mouse monoclonal 1:5000; Santa Cruz Biotechnology).

    Techniques: In Vivo, In Vitro, Staining, Knock-Out, Immunostaining, Serial Dilution, Software, Real-time Polymerase Chain Reaction

    Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P

    Journal: Nature Communications

    Article Title: Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

    doi: 10.1038/s41467-017-02720-9

    Figure Lengend Snippet: Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower right) Quantification of the proportion of Tuj1 + or MAP2 + cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67 + ( n = 2 or 3) or Nestin + ( n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF ( n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP + cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d ( n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting ( n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f . DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (* P

    Article Snippet: Antibodies and reagents Antibodies used in this study were anti-Klf4 (rabbit polyclonal 1:500; Gene Tex, Irvine, CA, USA), anti-Flag (mouse, 1:1000; Sigma, St. Louis, MO, USA), anti-HA (rabbit, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Stau1 (rabbit, 1:500; Thermo Scientific, Rockford, IL, USA), anti-Map2 (rabbit polyclonal 1:200, Chemicon, Temecula, CA, USA), anti-Nestin (mouse monoclonal 1:350, BD Biosciences, San Jose, CA, USA), anti-Tuj1 (mouse polyclonal 1:200; Covance, Princeton, NJ, USA), anti-Tbr1 (rabbit polyclonal 1:200; Abcam Ltd, Cambridge, MA, USA), anti-Pax6 (rabbit polyclonal 1:200; Abcam Ltd), anti-Ddx5 (rabbit polyclonal 1:1000; Abcam Ltd), anti-Ddx17 (rabbit polyclonal 1:1000; Abcam Ltd), anti-GST (rabbit polyclonal 1:500; Santa Cruz Biotechnology), anti-BrdU (mouse monoclonal 1:500; Abcam Ltd), anti-Dlx1 (mouse monoclonal 1:250; Abnova, Walnut, CA, USA), anti-Dlx2 (rabbit polyclonal, 1:250; Abcam Ltd), anti-Ctip2 (Rat 1:200; Abcam Ltd), anti-cleaved caspase-3 (mouse 1:200; R & D Systems, Inc., Minneapolis, MN, USA), anti-UPF1 (rabbit 1:200; Cell Signaling Technology Inc., Danvers, MA, USA), and anti-α-tubulin (mouse monoclonal 1:5000; Santa Cruz Biotechnology).

    Techniques: Immunostaining, Infection, Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

    Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n = 2). See also Supplementary Fig. 8a . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P

    Journal: Nature Communications

    Article Title: Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis

    doi: 10.1038/s41467-017-02720-9

    Figure Lengend Snippet: Stau1 inhibits neurogenesis in a Klf4-dependent manner. a (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 (#1 and #3) lentivirus. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1, Stau2, and α-tubulin in samples analyzed in a were were detected by immunoblotting ( n = 2). See also Supplementary Fig. 8a . (Lower right) Corresponding quantification of number of Tuj1 + and Map2 + cells. b (Upper) Immunostaining with Nestin or Ki67 (both red) antibodies of samples analyzed in a . DAPI (blue). Scale bars, 50 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + ( n = 2 or 3) cells. c (Upper) Immunostaining of NPCs infected with pCDH-control or pCDH-Stau1 lentivirus with Tuj1 (green) or MAP2 (red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower left) Stau1 and α-tubulin in samples analyzed in c were detected by immunoblotting ( n = 2). (Lower) Corresponding quantification of data shown above. d (Upper) Immunostaining of samples equivalent to those shown in c with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 25 μm. (Lower) Corresponding quantification of Nestin + ( n = 3) and Ki67 + (Ctrl, n = 3; Stau1, n = 2) cells. e RT-qPCR of indicated transcripts in control- or Klf4-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shStau1 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). f RT-qPCR of indicated transcripts in control- or Stau1-overexpressing NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 lentivirus cultured for 0.2 or 2 days in differentiation conditions ( n = 3). Data are shown as mean ± SD. ANOVA tests were performed to calculate statistical significance (* P

    Article Snippet: Antibodies and reagents Antibodies used in this study were anti-Klf4 (rabbit polyclonal 1:500; Gene Tex, Irvine, CA, USA), anti-Flag (mouse, 1:1000; Sigma, St. Louis, MO, USA), anti-HA (rabbit, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Stau1 (rabbit, 1:500; Thermo Scientific, Rockford, IL, USA), anti-Map2 (rabbit polyclonal 1:200, Chemicon, Temecula, CA, USA), anti-Nestin (mouse monoclonal 1:350, BD Biosciences, San Jose, CA, USA), anti-Tuj1 (mouse polyclonal 1:200; Covance, Princeton, NJ, USA), anti-Tbr1 (rabbit polyclonal 1:200; Abcam Ltd, Cambridge, MA, USA), anti-Pax6 (rabbit polyclonal 1:200; Abcam Ltd), anti-Ddx5 (rabbit polyclonal 1:1000; Abcam Ltd), anti-Ddx17 (rabbit polyclonal 1:1000; Abcam Ltd), anti-GST (rabbit polyclonal 1:500; Santa Cruz Biotechnology), anti-BrdU (mouse monoclonal 1:500; Abcam Ltd), anti-Dlx1 (mouse monoclonal 1:250; Abnova, Walnut, CA, USA), anti-Dlx2 (rabbit polyclonal, 1:250; Abcam Ltd), anti-Ctip2 (Rat 1:200; Abcam Ltd), anti-cleaved caspase-3 (mouse 1:200; R & D Systems, Inc., Minneapolis, MN, USA), anti-UPF1 (rabbit 1:200; Cell Signaling Technology Inc., Danvers, MA, USA), and anti-α-tubulin (mouse monoclonal 1:5000; Santa Cruz Biotechnology).

    Techniques: Immunostaining, Infection, Quantitative RT-PCR, Cell Culture

    Generation of NMR-iPSCs from adult fibroblasts. ( a ) Adult NMR. ( b ) Morphology of NMR-fibroblasts. ( c ) NMR-iPSCs (clone 27). ( d ) AP activity. ( e ) Karyotype of NMR-iPSCs (clone 24) at passage 10. ( f ) RNA-seq of expression levels of selected pluripotency and fibroblast markers in NMR-iPSCs and NMR-fibroblasts. Y axis: ratio of the average value of fragments per kilobase of transcript per million mapped reads (FPKM) of four NMR-iPSC clones to the average of NMR-fibroblast lines. ( g ) Immunocytochemical analyses of the expression of differentiated EBs is shown as follows: mesoderm (DESMIN, α-smooth muscle actin (αSMA)), endoderm (FOXA2 and VIMENTIN) and ectoderm (NESTIN and GFAP) markers. ( h ) Tumours or testes after transplantation of human-iPSCs (10 weeks), Ms-iPSCs (4 weeks) or NMR-iPSCs (10 or 20 weeks) into the testes of NOD/SCID mice. ( i ) Weights of tumours and testes. Ten weeks ( n =20), 20 weeks ( n =16) or 28 weeks ( n =10) for NMR; 10 weeks ( n =8) for human, 4 weeks ( n =8) for mouse. n : transplanted testes. Y axis: weights in 6+log 2 arbitrary units. The data are represented as mean±s.e.m. * P

    Journal: Nature Communications

    Article Title: Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

    doi: 10.1038/ncomms11471

    Figure Lengend Snippet: Generation of NMR-iPSCs from adult fibroblasts. ( a ) Adult NMR. ( b ) Morphology of NMR-fibroblasts. ( c ) NMR-iPSCs (clone 27). ( d ) AP activity. ( e ) Karyotype of NMR-iPSCs (clone 24) at passage 10. ( f ) RNA-seq of expression levels of selected pluripotency and fibroblast markers in NMR-iPSCs and NMR-fibroblasts. Y axis: ratio of the average value of fragments per kilobase of transcript per million mapped reads (FPKM) of four NMR-iPSC clones to the average of NMR-fibroblast lines. ( g ) Immunocytochemical analyses of the expression of differentiated EBs is shown as follows: mesoderm (DESMIN, α-smooth muscle actin (αSMA)), endoderm (FOXA2 and VIMENTIN) and ectoderm (NESTIN and GFAP) markers. ( h ) Tumours or testes after transplantation of human-iPSCs (10 weeks), Ms-iPSCs (4 weeks) or NMR-iPSCs (10 or 20 weeks) into the testes of NOD/SCID mice. ( i ) Weights of tumours and testes. Ten weeks ( n =20), 20 weeks ( n =16) or 28 weeks ( n =10) for NMR; 10 weeks ( n =8) for human, 4 weeks ( n =8) for mouse. n : transplanted testes. Y axis: weights in 6+log 2 arbitrary units. The data are represented as mean±s.e.m. * P

    Article Snippet: For immunocytochemical analyses, the cells were fixed with 4% paraformaldehyde and analysed using the antibodies against the proteins as follows: αSMA (Sigma Aldrich; 1A4; 1:1,000), albumin (Sigma Aldrich; A0433; 1:1,000), vimentin (Abcam; EPR3776; 1:1,000), Nestin (BD Pharmingen; BD-556309; 1:250), Tubb3 (Sigma Aldrich; T8660; 1:1,000).

    Techniques: Nuclear Magnetic Resonance, Activity Assay, RNA Sequencing Assay, Expressing, Clone Assay, Transplantation Assay, Mass Spectrometry, Mouse Assay

    Abnormal development of neural stem cell-derived neurospheres from Raldh2 −/− spinal cords. Neurospheres were derived from dorsal cervical/brachial spinal cord from E12.5 WT and Raldh2 −/− embryos. (A,B) After growth of dissociated progenitor cells in suspension, in 6-well plates, for 10 days in a defined selective medium (see Materials and Methods ), the number of spheres developing from Raldh2 −/− embryos spinal cords is decreased by 25% compared to WT (E: 11.1±0.88 spheres/well for WT; 8.40±1.50 for mutants; n = 9; T- test: P value = 0,0009). (C,D) To further study progenitor cell differentiation, spheres were plated onto laminin-coated coverslips for 3 days, after 10 days of growth in suspension. Raldh2 −/− derived spheres exhibited reduced number of cells compared to WT (F: one quadrant of a plated WT sphere is composed of 105.4±16.6 cells, against 40.9±6,82 cells in a Raldh2 −/− sphere; DAPI positive nuclei were counted using ImageJ software; n = 9; P = 1.6×10 −5 ). (G–O) After growth onto laminin coated coverslips for 3 days, cells were processed for immunocytochemistry. Nestin+ cells (I,M) are increased by 20% in the Raldh2 −/− derived spheres, while TuJ1+ cells (J,N) are decreased by half. (H,L, DAPI staining; K,O, merged images). (G) After counting and normalization for total cell numbers, assessed by DAPI positive cell nuclei, one quadrant of a WT sphere contains 41.6±14 Nestin+ and 42.0±9.76 TuJ1+ cells, against 57.9±9.21 and 25.9±6,66 in a Raldh2 −/− sphere (n = 9; P = 0,001 and 0,007, respectively).

    Journal: PLoS ONE

    Article Title: Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

    doi: 10.1371/journal.pone.0032447

    Figure Lengend Snippet: Abnormal development of neural stem cell-derived neurospheres from Raldh2 −/− spinal cords. Neurospheres were derived from dorsal cervical/brachial spinal cord from E12.5 WT and Raldh2 −/− embryos. (A,B) After growth of dissociated progenitor cells in suspension, in 6-well plates, for 10 days in a defined selective medium (see Materials and Methods ), the number of spheres developing from Raldh2 −/− embryos spinal cords is decreased by 25% compared to WT (E: 11.1±0.88 spheres/well for WT; 8.40±1.50 for mutants; n = 9; T- test: P value = 0,0009). (C,D) To further study progenitor cell differentiation, spheres were plated onto laminin-coated coverslips for 3 days, after 10 days of growth in suspension. Raldh2 −/− derived spheres exhibited reduced number of cells compared to WT (F: one quadrant of a plated WT sphere is composed of 105.4±16.6 cells, against 40.9±6,82 cells in a Raldh2 −/− sphere; DAPI positive nuclei were counted using ImageJ software; n = 9; P = 1.6×10 −5 ). (G–O) After growth onto laminin coated coverslips for 3 days, cells were processed for immunocytochemistry. Nestin+ cells (I,M) are increased by 20% in the Raldh2 −/− derived spheres, while TuJ1+ cells (J,N) are decreased by half. (H,L, DAPI staining; K,O, merged images). (G) After counting and normalization for total cell numbers, assessed by DAPI positive cell nuclei, one quadrant of a WT sphere contains 41.6±14 Nestin+ and 42.0±9.76 TuJ1+ cells, against 57.9±9.21 and 25.9±6,66 in a Raldh2 −/− sphere (n = 9; P = 0,001 and 0,007, respectively).

    Article Snippet: Anti-β-III-tubulin (TuJ1, Covance) and anti-Nestin (rat-401, Developmental Studies Hybridoma Bank) were used at 1/600 and 1/100 dilutions, respectively.

    Techniques: Derivative Assay, Cell Differentiation, Software, Immunocytochemistry, Staining

    Mitotic neuroblasts in the embryonic mouse cortex with lagging chromosomes. ( A and B ) Low magnification (×20) micrographs of the embryonic cerebral cortex. Immunofluorescence for the intermediate filament protein nestin ( A ), a neural progenitor cell marker, illustrates the distribution of neuroblasts in the ventricular zone (VZ) of the embryonic cerebral cortex. Phospho-H3 labeling ( B , red) reveals mitotic neuroblasts concentrated at the ventricular surface (bottom) of the VZ. Nuclei are counterstained with DAPI (blue). ( C – F ) High magnification (×100) Z stacks from deconvolution microscopy of phospho-H3-labeled mitotic figures at the bottom of the VZ reveal morphologically normal prometaphase/metaphase ( C ) and anaphase ( D ) profiles. In addition, lagging chromosomes (arrows) are readily observed in prometaphase/metaphase ( E ) and anaphase ( F ) profiles.

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

    Article Title: Chromosomal variation in neurons of the developing and adult mammalian nervous system

    doi: 10.1073/pnas.231487398

    Figure Lengend Snippet: Mitotic neuroblasts in the embryonic mouse cortex with lagging chromosomes. ( A and B ) Low magnification (×20) micrographs of the embryonic cerebral cortex. Immunofluorescence for the intermediate filament protein nestin ( A ), a neural progenitor cell marker, illustrates the distribution of neuroblasts in the ventricular zone (VZ) of the embryonic cerebral cortex. Phospho-H3 labeling ( B , red) reveals mitotic neuroblasts concentrated at the ventricular surface (bottom) of the VZ. Nuclei are counterstained with DAPI (blue). ( C – F ) High magnification (×100) Z stacks from deconvolution microscopy of phospho-H3-labeled mitotic figures at the bottom of the VZ reveal morphologically normal prometaphase/metaphase ( C ) and anaphase ( D ) profiles. In addition, lagging chromosomes (arrows) are readily observed in prometaphase/metaphase ( E ) and anaphase ( F ) profiles.

    Article Snippet: The primary anti-nestin (PharMingen) and anti-phosphorylated histone H3 (phospho-H3; Upstate Biotechnology, Lake Placid, NY) Abs were detected with a Cy3-conjugated secondary IgG (Jackson ImmunoResearch).

    Techniques: Immunofluorescence, Marker, Labeling, Microscopy

    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

    PATZ1 localization in the stem cell compartment of GBM Representative confocal microscopy images of GBM (two samples with high PATZ1 score (+++) and GBM-associated normal tissues stained with anti-NESTIN (A, E) , anti-OCT-4 (I) and anti-PATZ1 (B, F, J) . (D, H, L) Merge images showing co-localization of PATZ1 with either NESTIN or OCT-4. Scale bar: 20 μm. (C, G, K) Nuclei are depicted by the blue fluorescence of DAPI.

    Journal: Oncotarget

    Article Title: PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype

    doi: 10.18632/oncotarget.19546

    Figure Lengend Snippet: PATZ1 localization in the stem cell compartment of GBM Representative confocal microscopy images of GBM (two samples with high PATZ1 score (+++) and GBM-associated normal tissues stained with anti-NESTIN (A, E) , anti-OCT-4 (I) and anti-PATZ1 (B, F, J) . (D, H, L) Merge images showing co-localization of PATZ1 with either NESTIN or OCT-4. Scale bar: 20 μm. (C, G, K) Nuclei are depicted by the blue fluorescence of DAPI.

    Article Snippet: The primary antibodies used were: anti-PATZ1 [ ]; anti-NESTIN (sc-23927, Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDGFRβ (3169, Cell Signaling Technology, Boston, MA); anti-NANOG (sc-134218, Santa Cruz Biotechnology); anti-SOX2 (sc-20088, Santa Cruz Biotechnology); anti-CXCR4 (C8727, Sigma-Aldrich).

    Techniques: Confocal Microscopy, Staining, Fluorescence

    PATZ1 expression in GSC cultures (A) Representative phase contrast pictures of human glioma cells cultured in media selective (GSC, right panels) or not (DIFF, left panels) for the growth of glioma stem cells. The upper-right panel displays GSC growing as neurosphere, while the lower-right panel displays GSC growing in adhesion. Scale bar: 100μm. (B) Gene expression changes of PATZ1 in human glioma cells grown in media selective (GSC) or not (DIFF) for the growth of GSC, as assessed by qRT-PCR. Data are presented as mean ± SD. (C) Gene expression changes of PATZ1 in GSC growing in adhesion (adherent) or in suspension (sphere), as assessed by qRT-PCR. Data are presented as mean ± SD. (D) Representative immunofluorescence images depicting in yellow the expression of OCT4, NANOG, SOX-2, and NESTIN in GSC growing either in adhesion (GSC adherent, lower panels) or in suspensions (GSC sphere, upper panels). Nuclei are depicted by the blue fluorescence of DAPI. Scale bar: 20 μm. (E) Representative confocal immunofluorescence image (merge image) showing the expression of PATZ1 (yellow fluorescence) and Nestin (red fluorescence) in GSCs. Nuclei are depicted by the blue fluorescence of DAPI. Single immunodetection of either PATZ1 or Nestin in the inset area, and digital merge of the images, are shown in the middle. Negative control obtained by omitting the primary antibodies is shown on the right. Scale bar: 20 μm.

    Journal: Oncotarget

    Article Title: PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype

    doi: 10.18632/oncotarget.19546

    Figure Lengend Snippet: PATZ1 expression in GSC cultures (A) Representative phase contrast pictures of human glioma cells cultured in media selective (GSC, right panels) or not (DIFF, left panels) for the growth of glioma stem cells. The upper-right panel displays GSC growing as neurosphere, while the lower-right panel displays GSC growing in adhesion. Scale bar: 100μm. (B) Gene expression changes of PATZ1 in human glioma cells grown in media selective (GSC) or not (DIFF) for the growth of GSC, as assessed by qRT-PCR. Data are presented as mean ± SD. (C) Gene expression changes of PATZ1 in GSC growing in adhesion (adherent) or in suspension (sphere), as assessed by qRT-PCR. Data are presented as mean ± SD. (D) Representative immunofluorescence images depicting in yellow the expression of OCT4, NANOG, SOX-2, and NESTIN in GSC growing either in adhesion (GSC adherent, lower panels) or in suspensions (GSC sphere, upper panels). Nuclei are depicted by the blue fluorescence of DAPI. Scale bar: 20 μm. (E) Representative confocal immunofluorescence image (merge image) showing the expression of PATZ1 (yellow fluorescence) and Nestin (red fluorescence) in GSCs. Nuclei are depicted by the blue fluorescence of DAPI. Single immunodetection of either PATZ1 or Nestin in the inset area, and digital merge of the images, are shown in the middle. Negative control obtained by omitting the primary antibodies is shown on the right. Scale bar: 20 μm.

    Article Snippet: The primary antibodies used were: anti-PATZ1 [ ]; anti-NESTIN (sc-23927, Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDGFRβ (3169, Cell Signaling Technology, Boston, MA); anti-NANOG (sc-134218, Santa Cruz Biotechnology); anti-SOX2 (sc-20088, Santa Cruz Biotechnology); anti-CXCR4 (C8727, Sigma-Aldrich).

    Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Immunodetection, Negative Control

    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

    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

    KLF4 overexpression blocks GSC differentiation by RA/FBS in-vitro . ( a ) Stable GSC cell-lines carrying a viral-transduced KLF4 expression vector or control (empty vector), were subjected to in-vitro differentiation using RA/FBS. Cells were immunostained for GFAP (a marker of differentiation) and Nestin (a marker of the undifferentiated state) in both NBE and RA/FBS conditions after 10 days. KLF4 overexpressing GSC lines show both lower GFAP and higher Nestin staining intensity after 10-day incubation with RA/FBS media compared to the control. ( b ) Stably transfected GSCs carrying a doxycycline-inducible vector retained nestin expression and lost GFAP expression intensity in the presence of doxycycline (KLF4 induction), as observed via western blot. The SRA condition indicates differentiating conditions by RA/FBS (Images are cropped from the original, see). ( c ) Overexpression of KLF4 decreases proliferation of glioblastoma stem cells relative to a control (p

    Journal: Scientific Reports

    Article Title: A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity

    doi: 10.1038/srep43605

    Figure Lengend Snippet: KLF4 overexpression blocks GSC differentiation by RA/FBS in-vitro . ( a ) Stable GSC cell-lines carrying a viral-transduced KLF4 expression vector or control (empty vector), were subjected to in-vitro differentiation using RA/FBS. Cells were immunostained for GFAP (a marker of differentiation) and Nestin (a marker of the undifferentiated state) in both NBE and RA/FBS conditions after 10 days. KLF4 overexpressing GSC lines show both lower GFAP and higher Nestin staining intensity after 10-day incubation with RA/FBS media compared to the control. ( b ) Stably transfected GSCs carrying a doxycycline-inducible vector retained nestin expression and lost GFAP expression intensity in the presence of doxycycline (KLF4 induction), as observed via western blot. The SRA condition indicates differentiating conditions by RA/FBS (Images are cropped from the original, see). ( c ) Overexpression of KLF4 decreases proliferation of glioblastoma stem cells relative to a control (p

    Article Snippet: Western blots were performed with the following antibodies: anti-beta actin (Sigma), anti-EGR1 and anti-Hes1 (Santa Cruz), anti-GFAP and anti-Histone H3, anti-KLF4, anti-SOX2 (R & D), anti-nestin (Covance), anti-beta-tubulin (Sigma).

    Techniques: Over Expression, In Vitro, Expressing, Plasmid Preparation, Marker, Staining, Incubation, Stable Transfection, Transfection, Western Blot

    Nestin and CD15 expression after in vitro treatment with TMZ, Dox and a combination of both drugs. Upper panels show western blot analysis of GBM non-CSCs under different treatment conditions (50μM TMZ, 50μM Dox or 50μM each), Tubulin represents the loading control. Lower panels are results from densitometric scanning analyses of the western blot signals. Results are given as relative expression to untreated control cells.

    Journal: PLoS ONE

    Article Title: Temozolomide-induced increase of tumorigenicity can be diminished by targeting of mitochondria in in vitro models of patient individual glioblastoma

    doi: 10.1371/journal.pone.0191511

    Figure Lengend Snippet: Nestin and CD15 expression after in vitro treatment with TMZ, Dox and a combination of both drugs. Upper panels show western blot analysis of GBM non-CSCs under different treatment conditions (50μM TMZ, 50μM Dox or 50μM each), Tubulin represents the loading control. Lower panels are results from densitometric scanning analyses of the western blot signals. Results are given as relative expression to untreated control cells.

    Article Snippet: Immunohistochemistry (IHC) was performed using antibodies specific for nestin (Merck Millipore) and CD15 (immunotools).

    Techniques: Expressing, In Vitro, Western Blot

    CD15 expression is increased in clinical samples of two relapsed GBM. IHC staining of nestin and CD15 in clinical samples of two cases pre and post chemotherapy with TMZ, 200x magnification.

    Journal: PLoS ONE

    Article Title: Temozolomide-induced increase of tumorigenicity can be diminished by targeting of mitochondria in in vitro models of patient individual glioblastoma

    doi: 10.1371/journal.pone.0191511

    Figure Lengend Snippet: CD15 expression is increased in clinical samples of two relapsed GBM. IHC staining of nestin and CD15 in clinical samples of two cases pre and post chemotherapy with TMZ, 200x magnification.

    Article Snippet: Immunohistochemistry (IHC) was performed using antibodies specific for nestin (Merck Millipore) and CD15 (immunotools).

    Techniques: Expressing, Immunohistochemistry, Staining

    Immunohistochemical staining of neuron-like cells in treatment groups for NSE, nestin, and GFAP, respectively. The arrows in figures indicate neuron-like cells.

    Journal: Stem Cells International

    Article Title: Synaptic Plasticity of Human Umbilical Cord Mesenchymal Stem Cell Differentiating into Neuron-like Cells In Vitro Induced by Edaravone

    doi: 10.1155/2018/5304279

    Figure Lengend Snippet: Immunohistochemical staining of neuron-like cells in treatment groups for NSE, nestin, and GFAP, respectively. The arrows in figures indicate neuron-like cells.

    Article Snippet: LLC., St. Louis, MO 63178,USA); FITC-CD19, FITC-CD34, PE-CD11b, PE-CD73, PE-CD90, PE-CD45, and PE-CD105 (Becton, Dickinson and Company, Franklin Lakes, New Jersey 07417-1880); PS immunohistochemistry kit (Beijing Zhongshan Golden Bridge Company, China); Taq PCR star mix (Genstar, China); EasyScript First-Strand cDNA synthesis supermix (TransGen Biotech, China); TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa Japan); cell RIPA Lysis Buffer and phenylmethylsulfonyl fluoride (Solarbio, Beijing, China); polyclonal antibodies of neuron-specific enolase (NSE) and nestin (OriGene, USA); glial fibrillary acidic protein (GFAP) and glyceraldehyde-3-phosphate dehydrogenase (Affbiotech, USA); and polyclonal antibodies of synaptophysin, growth-associated protein 43, and postsynaptic density 95 (Abways, China).

    Techniques: Immunohistochemistry, Staining

    Characterization of spontaneous mitochondrial SO flashes in NPCs. (A): Dissociated NPCs at day 3 in culture were immunostained with antibodies against Sox2, nestin and BrdU (after a 16 h exposure to 10 μM BrdU) (green), which are markers of proliferating

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Mitochondrial Superoxide Production Negatively Regulates Neural Progenitor Proliferation and Cerebral Cortical Development

    doi: 10.1002/stem.1213

    Figure Lengend Snippet: Characterization of spontaneous mitochondrial SO flashes in NPCs. (A): Dissociated NPCs at day 3 in culture were immunostained with antibodies against Sox2, nestin and BrdU (after a 16 h exposure to 10 μM BrdU) (green), which are markers of proliferating

    Article Snippet: NPCs or brain sections were processed for immunocytochemistry using the following primary antibodies and dilutions: anti-ß3 -tubulin (Tuj1; mouse, 1:250; Sigma, St. Louis, MO), anti-Sox2 (1:200, Chemicon, Temecula, CA), anti-nestin (1:200, Cell Signaling Technology, Danvers, MA), anti Ki67 (1:200, Chemicon, Temecula, CA, ).

    Techniques: