rabbit anti sox2  (Millipore)


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    Structured Review

    Millipore rabbit anti sox2
    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for <t>SOX2</t> and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows
    Rabbit Anti Sox2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sox2/product/Millipore
    Average 99 stars, based on 94 article reviews
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    rabbit anti sox2 - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate"

    Article Title: Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.065870

    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for SOX2 and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows
    Figure Legend Snippet: Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for SOX2 and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows

    Techniques Used: Expressing, Immunofluorescence

    Increased Sus cells in Gdf11 –/– and ActβB –/– ;Gdf11 –/– OE. ( A ) ISH with the indicated probes indicates increased Sox2 expression and a thicker Sus layer (white arrowhead) in Act β B –/–
    Figure Legend Snippet: Increased Sus cells in Gdf11 –/– and ActβB –/– ;Gdf11 –/– OE. ( A ) ISH with the indicated probes indicates increased Sox2 expression and a thicker Sus layer (white arrowhead) in Act β B –/–

    Techniques Used: In Situ Hybridization, Expressing, Activated Clotting Time Assay

    Increase in SOX2 + and ASCL1 + cells in ActβB –/– OE. Bar charts show cells/mm OE (mean ± s.e.m.; * , P ≤0.05 DT). White asterisk, Bowman's gland. BL, basal lamina. Scale bars: 20 μm.
    Figure Legend Snippet: Increase in SOX2 + and ASCL1 + cells in ActβB –/– OE. Bar charts show cells/mm OE (mean ± s.e.m.; * , P ≤0.05 DT). White asterisk, Bowman's gland. BL, basal lamina. Scale bars: 20 μm.

    Techniques Used:

    ActβB and GDF11 modulate stem cell fates. ( A-C ) Cells in three marker categories (SOX2 + , black; ASCL1 + , white; SOX2 + ; ASCL1 + , gray) were quantified for (A) total OE, (B) basal stem/progenitor cell compartment and (C) apical sustentacular cell
    Figure Legend Snippet: ActβB and GDF11 modulate stem cell fates. ( A-C ) Cells in three marker categories (SOX2 + , black; ASCL1 + , white; SOX2 + ; ASCL1 + , gray) were quantified for (A) total OE, (B) basal stem/progenitor cell compartment and (C) apical sustentacular cell

    Techniques Used: Marker

    2) Product Images from "Derivation and Long-Term Culture of an Embryonic Stem Cell-Like Line from Zebrafish Blastomeres Under Feeder-Free Condition"

    Article Title: Derivation and Long-Term Culture of an Embryonic Stem Cell-Like Line from Zebrafish Blastomeres Under Feeder-Free Condition

    Journal: Zebrafish

    doi: 10.1089/zeb.2013.0879

    Comparative expression of sox2, nanog, lin28 , and pou5f1 in ZES1 and zebrafish adult liver cell line ZFL using semi-quantitative real-time PCR. Relative expression levels were based on normalization to β-actin level. Results shown here are representative
    Figure Legend Snippet: Comparative expression of sox2, nanog, lin28 , and pou5f1 in ZES1 and zebrafish adult liver cell line ZFL using semi-quantitative real-time PCR. Relative expression levels were based on normalization to β-actin level. Results shown here are representative

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Overlapped histogram of SOX2 expression in ZES1 cells as analyzed by fluorescent-activated cell sorting. R1=ZES1 immunostained with FITC secondary antibody only, R2=ZES1 immunostained with anti-SOX2 primary antibody, and FITC secondary antibody. SOX2-positive
    Figure Legend Snippet: Overlapped histogram of SOX2 expression in ZES1 cells as analyzed by fluorescent-activated cell sorting. R1=ZES1 immunostained with FITC secondary antibody only, R2=ZES1 immunostained with anti-SOX2 primary antibody, and FITC secondary antibody. SOX2-positive

    Techniques Used: Expressing, FACS

    Immunostaining of ZES1 cells and differentiated ZES1 cells with SOX2. Undifferentiated ZES1 cells exhibited positive staining with (A) SOX2 and (B) DAPI. RA-treated ZES1 cells also showed positive (C) SOX2 staining at neural structures and (D) DAPI at
    Figure Legend Snippet: Immunostaining of ZES1 cells and differentiated ZES1 cells with SOX2. Undifferentiated ZES1 cells exhibited positive staining with (A) SOX2 and (B) DAPI. RA-treated ZES1 cells also showed positive (C) SOX2 staining at neural structures and (D) DAPI at

    Techniques Used: Immunostaining, Staining

    3) Product Images from "Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo"

    Article Title: Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3975-14.2015

    Expression analysis of molecular markers of iN cells. A–H , mCherry + cells in the dorsal midbrain derived from WT mice that were infected with the virus AAV–Ascl1/mCherry at P12–P15 were collected by FACS on day 4 (D4, black bars), day 10 (D10, red bars), and day 30 (D30, blue bars) after infection. The expression of the astrocyte markers Gfap ( A ), S100 β ( B ), and Acsbg1 ( C ), the neuronal markers Tuj1 ( D ), Map2 ( E ), and NeuN ( F ), the neural progenitor markers Sox2 ( G ) and Pax6 ( H ), and the midbrain neural progenitor markers En1 , En2 , Pax3 , and Pax7 ( I ) was examined by qRT-PCR. The neurospheres (yellow bars) derived from the SVZ of mice at P0 were used as positive controls for detecting the expression of Sox2 and Pax6 . The cells derived from E12.5 midbrain (purple bars) were used as positive controls for detecting the expression of En1 , En2 , Pax3 , and Pax7 .
    Figure Legend Snippet: Expression analysis of molecular markers of iN cells. A–H , mCherry + cells in the dorsal midbrain derived from WT mice that were infected with the virus AAV–Ascl1/mCherry at P12–P15 were collected by FACS on day 4 (D4, black bars), day 10 (D10, red bars), and day 30 (D30, blue bars) after infection. The expression of the astrocyte markers Gfap ( A ), S100 β ( B ), and Acsbg1 ( C ), the neuronal markers Tuj1 ( D ), Map2 ( E ), and NeuN ( F ), the neural progenitor markers Sox2 ( G ) and Pax6 ( H ), and the midbrain neural progenitor markers En1 , En2 , Pax3 , and Pax7 ( I ) was examined by qRT-PCR. The neurospheres (yellow bars) derived from the SVZ of mice at P0 were used as positive controls for detecting the expression of Sox2 and Pax6 . The cells derived from E12.5 midbrain (purple bars) were used as positive controls for detecting the expression of En1 , En2 , Pax3 , and Pax7 .

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Infection, FACS, Quantitative RT-PCR

    4) Product Images from "SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium"

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2959

    Evidence for MIR450 cluster as a potential repressor of SOX2. (A): Schematic representation of the 3′UTR of human SOX2 and predicted binding site of miR‐450a and miR‐450b identified by TargetScan. (B): Sequence and complementation of the predicted binding site. (C): Illustration of the human MIR450 cluster (defined by miRBase) that includes six miRNAs genes. (D): Human embryonic stem cells were seeded on collagen IV‐coated dishes in the present of corneal fibroblast conditional media to induce corneal epithelial differentiation for the indicated time. Relative expression of the indicated miRNAs is shown and data represent the normalized expression as fold change in expression relative to undifferentiated cells. (E): Wholemount in situ hybridization for miR‐450b on mouse embryos of the indicated embryonic day. Increased magnifications are shown from left to right, and lens is annotated by white arrowheads. (F): Immunofluorescence staining of SOX2 on mouse head sections at E10.5 and E11.5. Nuclei were counterstained with DAPI. (G, H): In situ hybridization of miR‐450b on whole cornea (G) or sections of cornea (H) of 2‐month‐old mice. Scale bars are 250 μm (E, G) and 25 μm (F, H) . Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; lp, lens pit; lv, lens vesicle; oc, optic cup; pce, presumptive corneal epithelium; UTR, untranslated region.
    Figure Legend Snippet: Evidence for MIR450 cluster as a potential repressor of SOX2. (A): Schematic representation of the 3′UTR of human SOX2 and predicted binding site of miR‐450a and miR‐450b identified by TargetScan. (B): Sequence and complementation of the predicted binding site. (C): Illustration of the human MIR450 cluster (defined by miRBase) that includes six miRNAs genes. (D): Human embryonic stem cells were seeded on collagen IV‐coated dishes in the present of corneal fibroblast conditional media to induce corneal epithelial differentiation for the indicated time. Relative expression of the indicated miRNAs is shown and data represent the normalized expression as fold change in expression relative to undifferentiated cells. (E): Wholemount in situ hybridization for miR‐450b on mouse embryos of the indicated embryonic day. Increased magnifications are shown from left to right, and lens is annotated by white arrowheads. (F): Immunofluorescence staining of SOX2 on mouse head sections at E10.5 and E11.5. Nuclei were counterstained with DAPI. (G, H): In situ hybridization of miR‐450b on whole cornea (G) or sections of cornea (H) of 2‐month‐old mice. Scale bars are 250 μm (E, G) and 25 μm (F, H) . Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; lp, lens pit; lv, lens vesicle; oc, optic cup; pce, presumptive corneal epithelium; UTR, untranslated region.

    Techniques Used: Binding Assay, Sequencing, Expressing, In Situ Hybridization, Immunofluorescence, Staining, Mouse Assay

    SOX2 regulates long‐term colony‐forming efficiency and cell proliferation. Limbal cells were transfected with siSOX2 or siP63 or siCtl and 48 hours later, subjected to clonogenicity test as detailed in Materials and Methods section. Colonies were visualized by Rhodamin staining 3 weeks later (A, D) , and quantification of the number of colonies relative to control (B, E) and the average size of colonies (C, F) was performed by Nis‐Element software as detailed in Materials and Methods section. (G–I): Limbal stem/progenitor cells were transfected with siSOX2 or siCtl and 72 hours later, cells were immunostained for the proliferative marker Ki67 (G) , and quantification (by Nis‐Element software) of the relative number of Ki67‐positive cells is shown (H) . Transfectents were grown for 72 hours and then subjected to alamar blue viability test (I) . (B, C, E, F, H, I): Data represent mean ± SD, n = 3. Significance assessed by t test (*, p
    Figure Legend Snippet: SOX2 regulates long‐term colony‐forming efficiency and cell proliferation. Limbal cells were transfected with siSOX2 or siP63 or siCtl and 48 hours later, subjected to clonogenicity test as detailed in Materials and Methods section. Colonies were visualized by Rhodamin staining 3 weeks later (A, D) , and quantification of the number of colonies relative to control (B, E) and the average size of colonies (C, F) was performed by Nis‐Element software as detailed in Materials and Methods section. (G–I): Limbal stem/progenitor cells were transfected with siSOX2 or siCtl and 72 hours later, cells were immunostained for the proliferative marker Ki67 (G) , and quantification (by Nis‐Element software) of the relative number of Ki67‐positive cells is shown (H) . Transfectents were grown for 72 hours and then subjected to alamar blue viability test (I) . (B, C, E, F, H, I): Data represent mean ± SD, n = 3. Significance assessed by t test (*, p

    Techniques Used: Transfection, Staining, Software, Marker

    P63 rescues stemness in SOX2 knockdown cells. Limbal cells were co‐transfected with siSOX2 or siCtl and P63 expression plasmid or empty plasmid (veh). Seventy‐two hours later, cells were subjected to clonogenicity test (A) , and number of the colonies were quantified by Nis‐Element software as detailed in Materials and Methods section (B) . (C): Cells were taken for trypan blue assay to quantify dead cells after transfection of indicated factors. Quantitative real‐time polymerase chain reaction analysis of the indicated markers of stem/progenitor cells were performed (D) . (B–D): Data represents mean ± SD, n = 3. Statistical significance was assessed by one‐way analysis of varaince followed by Tukey′s test (*, p
    Figure Legend Snippet: P63 rescues stemness in SOX2 knockdown cells. Limbal cells were co‐transfected with siSOX2 or siCtl and P63 expression plasmid or empty plasmid (veh). Seventy‐two hours later, cells were subjected to clonogenicity test (A) , and number of the colonies were quantified by Nis‐Element software as detailed in Materials and Methods section (B) . (C): Cells were taken for trypan blue assay to quantify dead cells after transfection of indicated factors. Quantitative real‐time polymerase chain reaction analysis of the indicated markers of stem/progenitor cells were performed (D) . (B–D): Data represents mean ± SD, n = 3. Statistical significance was assessed by one‐way analysis of varaince followed by Tukey′s test (*, p

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

    miR‐450b represses SOX2 and induces differentiation of limbal epithelial stem/progenitor cells. (A): 293HEK cells were co‐transfected with SOX2‐3 ′ UTR luciferase plasmid or with a mutated plasmid with disrupted miR‐450a, b binding sites ( Mut‐SOX2‐3 ′ UTR , see Fig. S4 ), and with pre‐miR‐450a (PM450a) or pre‐miR‐450b‐5p (PM450b) or both or control (CtlPM), as indicated. Data represent the normalized luciferase activity relative to control sample. (B): Primary human limbal stem/progenitor cells were induced to differentiate for the indicated time and the expression of the indicated genes was examined by quantitative polymerase chain reaction. (C–J): Primary human limbal stem/progenitor cells were transfected with PM or AM or Ctl and then subjected to differentiation for 4 days and Western blot analysis of the indicated genes (C, G) , or transfectants were allowed to grow for 72 hours and then cell viability was tested by alamar blue assay (D, H) , or transfectants were subjected to clonogenicity test and colonies were revealed by rhodamine staining (E, I) and quantified (F, J) by Nis‐Element software. Data represent mean ± SD, n = 3. (A, B): Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test and (D, F, H, J) t test (*, p
    Figure Legend Snippet: miR‐450b represses SOX2 and induces differentiation of limbal epithelial stem/progenitor cells. (A): 293HEK cells were co‐transfected with SOX2‐3 ′ UTR luciferase plasmid or with a mutated plasmid with disrupted miR‐450a, b binding sites ( Mut‐SOX2‐3 ′ UTR , see Fig. S4 ), and with pre‐miR‐450a (PM450a) or pre‐miR‐450b‐5p (PM450b) or both or control (CtlPM), as indicated. Data represent the normalized luciferase activity relative to control sample. (B): Primary human limbal stem/progenitor cells were induced to differentiate for the indicated time and the expression of the indicated genes was examined by quantitative polymerase chain reaction. (C–J): Primary human limbal stem/progenitor cells were transfected with PM or AM or Ctl and then subjected to differentiation for 4 days and Western blot analysis of the indicated genes (C, G) , or transfectants were allowed to grow for 72 hours and then cell viability was tested by alamar blue assay (D, H) , or transfectants were subjected to clonogenicity test and colonies were revealed by rhodamine staining (E, I) and quantified (F, J) by Nis‐Element software. Data represent mean ± SD, n = 3. (A, B): Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test and (D, F, H, J) t test (*, p

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, CTL Assay, Western Blot, Alamar Blue Assay, Staining, Software

    SOX2 is co‐expressed with P63 in stem and progenitor cells of the corneal epithelium in vivo and in vitro. (A): Immunofluorescence staining of the indicated proteins was performed on paraffin sections of the adult mouse cornea. The regions of the limbus, peripheral cornea, and corneal center are shown. (B, C, E, F): Primary human limbal epithelial cells were differentiated for the indicated times, and the expression of the indicated marker was tested by quantitative real‐time polymerase chain reaction (qPCR) (B) or Western blot analysis (C) or immunostaining (E, F) . ERK served as loading control in (C) . (D): A comparative qPCR analysis of SOX2 in the following human cells: primary FE, LS, LE, iPSC, and ESCs. (B, D): Data were normalized to housekeeping gene and is presented (mean ± SD, n = 3) as fold increase compared to control sample. Statistical analysis was performed by one‐way analysis of variance followed by Tukey's test (*, p
    Figure Legend Snippet: SOX2 is co‐expressed with P63 in stem and progenitor cells of the corneal epithelium in vivo and in vitro. (A): Immunofluorescence staining of the indicated proteins was performed on paraffin sections of the adult mouse cornea. The regions of the limbus, peripheral cornea, and corneal center are shown. (B, C, E, F): Primary human limbal epithelial cells were differentiated for the indicated times, and the expression of the indicated marker was tested by quantitative real‐time polymerase chain reaction (qPCR) (B) or Western blot analysis (C) or immunostaining (E, F) . ERK served as loading control in (C) . (D): A comparative qPCR analysis of SOX2 in the following human cells: primary FE, LS, LE, iPSC, and ESCs. (B, D): Data were normalized to housekeeping gene and is presented (mean ± SD, n = 3) as fold increase compared to control sample. Statistical analysis was performed by one‐way analysis of variance followed by Tukey's test (*, p

    Techniques Used: In Vivo, In Vitro, Immunofluorescence, Staining, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining

    SOX2 can activate P63 enhancer and interact with P63 protein. (A, B): The sequence (A) and location (B) of C38 and C40 enhancers within P63 gene. Consensus binding sites of SOX2 and P63 are highlighted in pink and blue, respectively. (C): Schematic representation of luciferase construct containing C38, C40, C38‐C40, and C38‐C40‐mutated constructs lacking the indicated P63 or SOX2 binding sites. (D): HEK293 cells were co‐transfected with the indicated luciferase construct and with SOX2 or P63 or control empty plasmid (−), as indicated. Luciferase activity represents the relative read that was normalized to Renilla and presented as fold increase compared to control sample (mean ± SD, n = 3). Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test (*, p
    Figure Legend Snippet: SOX2 can activate P63 enhancer and interact with P63 protein. (A, B): The sequence (A) and location (B) of C38 and C40 enhancers within P63 gene. Consensus binding sites of SOX2 and P63 are highlighted in pink and blue, respectively. (C): Schematic representation of luciferase construct containing C38, C40, C38‐C40, and C38‐C40‐mutated constructs lacking the indicated P63 or SOX2 binding sites. (D): HEK293 cells were co‐transfected with the indicated luciferase construct and with SOX2 or P63 or control empty plasmid (−), as indicated. Luciferase activity represents the relative read that was normalized to Renilla and presented as fold increase compared to control sample (mean ± SD, n = 3). Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test (*, p

    Techniques Used: Sequencing, Binding Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay

    SOX2 prevents cell differentiation. Primary limbal cells were transfected with siSOX2 or control esiRNA, and 72 hours later, the expression of SOX2 and P63 was examined by real‐time polymerase chain reaction (PCR) (A) or Western blot analysis (B) . ERK served as loading control. Quantitative real‐time PCR analysis of the indicated markers of stem/progenitor cells (C) or markers of differentiated cells (D) , or cells were lysed and subjected to Western blot analysis of the indicated markers (E) (ERK served as loading control) or immunostaining of K3 was followed by flow cytometry analysis (F) . (G): The morphological changes upon siSOX2 repression are shown by bright field microscopy (×20 objective). (A, C, D): Data represent mean ± SD, n = 3 and statistical significance was assessed by t test (*, p
    Figure Legend Snippet: SOX2 prevents cell differentiation. Primary limbal cells were transfected with siSOX2 or control esiRNA, and 72 hours later, the expression of SOX2 and P63 was examined by real‐time polymerase chain reaction (PCR) (A) or Western blot analysis (B) . ERK served as loading control. Quantitative real‐time PCR analysis of the indicated markers of stem/progenitor cells (C) or markers of differentiated cells (D) , or cells were lysed and subjected to Western blot analysis of the indicated markers (E) (ERK served as loading control) or immunostaining of K3 was followed by flow cytometry analysis (F) . (G): The morphological changes upon siSOX2 repression are shown by bright field microscopy (×20 objective). (A, C, D): Data represent mean ± SD, n = 3 and statistical significance was assessed by t test (*, p

    Techniques Used: Cell Differentiation, Transfection, esiRNA, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Immunostaining, Flow Cytometry, Cytometry, Microscopy

    5) Product Images from "Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex"

    Article Title: Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex

    Journal: eNeuro

    doi: 10.1523/ENEURO.0219-18.2018

    Characterization of GFP-labeled NPC and cerebral organoids derived from hESC. A , Diagram of lentiviral vector expressing EGFP (enhanced GFP) driven by EF-1a promoter to label hES cells. B , Phase-contrast and fluorescent images of GFP-labeled hES cells (left) and expression of pluripotency markers Oct4 (green) and Nanog (red; right). C , Timeline of differentiation of hES cells into NPC. D , Phase-contrast and fluorescent images of human NPC derived from GFP-labeled hES cells. E , Representative immunofluorescence images of NPC stained for proliferation marker Ki67 and the indicated neural markers. F , Timeline of derivation of cerebral organoids from hESC. G , left panels, Phase-contrast and fluorescence images of GFP-labeled EB or Matrigel-embedded cerebral organoids. Right panel, Immunofluorescent images of sectioned cerebral organoids stained for GFP. H , Phase-contrast image of cerebral organoid at day 42 of culture and immunofluorescent images of sectioned day 42 cerebral organoid stained for the indicated markers. V: ventricle-like structures. Note the proliferative zone (Ki67+) and Sox2+ neuroprogenitors at the VZ/SVZ, and DCX+ neuroblasts in the outer layer.
    Figure Legend Snippet: Characterization of GFP-labeled NPC and cerebral organoids derived from hESC. A , Diagram of lentiviral vector expressing EGFP (enhanced GFP) driven by EF-1a promoter to label hES cells. B , Phase-contrast and fluorescent images of GFP-labeled hES cells (left) and expression of pluripotency markers Oct4 (green) and Nanog (red; right). C , Timeline of differentiation of hES cells into NPC. D , Phase-contrast and fluorescent images of human NPC derived from GFP-labeled hES cells. E , Representative immunofluorescence images of NPC stained for proliferation marker Ki67 and the indicated neural markers. F , Timeline of derivation of cerebral organoids from hESC. G , left panels, Phase-contrast and fluorescence images of GFP-labeled EB or Matrigel-embedded cerebral organoids. Right panel, Immunofluorescent images of sectioned cerebral organoids stained for GFP. H , Phase-contrast image of cerebral organoid at day 42 of culture and immunofluorescent images of sectioned day 42 cerebral organoid stained for the indicated markers. V: ventricle-like structures. Note the proliferative zone (Ki67+) and Sox2+ neuroprogenitors at the VZ/SVZ, and DCX+ neuroblasts in the outer layer.

    Techniques Used: Labeling, Derivative Assay, Plasmid Preparation, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

    Cell proliferation and NSC pool in cerebral organoid transplants. A , Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) stained for GFP and proliferation marker Ki67. D: dorsal, V: ventral, M: medial, L: lateral. B , Quantification (top) showing a higher density of Ki67+ cells per unit GFP+ area in cerebral organoid than in NPC transplants at both two and four weeks after transplantation. Bottom quantification: percentage of Ki67+/DAPI+ cells within the GFP+ cerebral organoid and NPC grafts. C , Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) showing abundant engrafted cells (GFP+) expressing stem cell marker Sox2 (red). White arrows: Sox2+ OPC in host cortical tissue. D , Quantification (top) showing no significant difference of the density of Sox2+ cells per unit GFP+ area between NPC and cerebral organoid transplants at either time points. Bottom quantification: percentage of Sox2+/DAPI+ cells within the organoid and NPC grafts showing no significant difference between the two types of transplants at either timepoint. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right; * p
    Figure Legend Snippet: Cell proliferation and NSC pool in cerebral organoid transplants. A , Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) stained for GFP and proliferation marker Ki67. D: dorsal, V: ventral, M: medial, L: lateral. B , Quantification (top) showing a higher density of Ki67+ cells per unit GFP+ area in cerebral organoid than in NPC transplants at both two and four weeks after transplantation. Bottom quantification: percentage of Ki67+/DAPI+ cells within the GFP+ cerebral organoid and NPC grafts. C , Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) showing abundant engrafted cells (GFP+) expressing stem cell marker Sox2 (red). White arrows: Sox2+ OPC in host cortical tissue. D , Quantification (top) showing no significant difference of the density of Sox2+ cells per unit GFP+ area between NPC and cerebral organoid transplants at either time points. Bottom quantification: percentage of Sox2+/DAPI+ cells within the organoid and NPC grafts showing no significant difference between the two types of transplants at either timepoint. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right; * p

    Techniques Used: Immunofluorescence, Staining, Marker, Transplantation Assay, Expressing

    6) Product Images from "Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells"

    Article Title: Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells

    Journal: Cell Cycle

    doi: 10.4161/cc.9.17.12792

    Factors involved in cytoplasmic responses to long dsRNAs in iPS cells and following differentiation of hESCs to trophoblasts. (A) Whole cell extracts were collected from IMR90 cells and iPS cells grown under feeder-free conditions and equal amounts of total proteins were subjected to SDS-PAGE and analyzed by western blotting with the indicated antibodies. Actin was used as a loading control and Sox2 was used as a marker for pluripotent cells. (B) Real-time RT-PCR was used to compare the relative levels of the mRNAs for the indicated factors between H9 cells and H9 cells that had been treated with BMP4 to promote differentiation to trophoblasts. Note that the bars represent the relative ratios of mRNA concentrations between the two cell populations (normalized to actin expression), not the absolute levels of transcripts. White bars, transcripts preferentially expressed in trophoblasts. Black bars, transcripts preferentially expressed in H9 cells. sox2, oct3/4 and lin28 , pluripotency markers. hcg β, trophoblast marker. (C) Real-time RT-PCR was used to compare the relative levels of the mRNAs for the indicated factors between IMR90 cells and iPS cells derived from IMR90 cells. Bars are as in (B). Another iPS cell line, iPS(IMR90)-2 showed essentially identical results (data not shown.)
    Figure Legend Snippet: Factors involved in cytoplasmic responses to long dsRNAs in iPS cells and following differentiation of hESCs to trophoblasts. (A) Whole cell extracts were collected from IMR90 cells and iPS cells grown under feeder-free conditions and equal amounts of total proteins were subjected to SDS-PAGE and analyzed by western blotting with the indicated antibodies. Actin was used as a loading control and Sox2 was used as a marker for pluripotent cells. (B) Real-time RT-PCR was used to compare the relative levels of the mRNAs for the indicated factors between H9 cells and H9 cells that had been treated with BMP4 to promote differentiation to trophoblasts. Note that the bars represent the relative ratios of mRNA concentrations between the two cell populations (normalized to actin expression), not the absolute levels of transcripts. White bars, transcripts preferentially expressed in trophoblasts. Black bars, transcripts preferentially expressed in H9 cells. sox2, oct3/4 and lin28 , pluripotency markers. hcg β, trophoblast marker. (C) Real-time RT-PCR was used to compare the relative levels of the mRNAs for the indicated factors between IMR90 cells and iPS cells derived from IMR90 cells. Bars are as in (B). Another iPS cell line, iPS(IMR90)-2 showed essentially identical results (data not shown.)

    Techniques Used: SDS Page, Western Blot, Marker, Quantitative RT-PCR, Expressing, Derivative Assay

    Some factors involved in cytoplasmic responses to long dsRNAs are expressed in hESCs. (A) Whole cell extracts were collected from HeLa cells and hESCs grown under feeder-free conditions and equal amounts of total proteins were subjected to SDS-PAGE and analyzed by western blotting with the indicated antibodies. Actin was used as a loading control and Sox2 was used as a marker for pluripotent cells. (B) Lack of dsRNA-induced PKR autophosphorylation in H9 cells. HeLa cells and H9 cells were transfected with 2 µg/mL PIC for 6 hrs and 24 hrs respectively, and whole cell extracts were collected from the untreated cells and the PIC treated cells at the indicated time points. Equal amounts of total proteins were loaded. Actin was used as a loading control.
    Figure Legend Snippet: Some factors involved in cytoplasmic responses to long dsRNAs are expressed in hESCs. (A) Whole cell extracts were collected from HeLa cells and hESCs grown under feeder-free conditions and equal amounts of total proteins were subjected to SDS-PAGE and analyzed by western blotting with the indicated antibodies. Actin was used as a loading control and Sox2 was used as a marker for pluripotent cells. (B) Lack of dsRNA-induced PKR autophosphorylation in H9 cells. HeLa cells and H9 cells were transfected with 2 µg/mL PIC for 6 hrs and 24 hrs respectively, and whole cell extracts were collected from the untreated cells and the PIC treated cells at the indicated time points. Equal amounts of total proteins were loaded. Actin was used as a loading control.

    Techniques Used: SDS Page, Western Blot, Marker, Transfection

    7) Product Images from "Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival"

    Article Title: Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3769-13.2014

    Ctcf -deficiency causes premature neurogenesis. A , Immunodetection of SOX2 (red) and TBR2 (green) in E14 control and Ctcf Nes-cre cortex. B , SOX2 + , TBR2 + , and SOX2 + /TBR2 + cells were quantified in 150-μm-wide cortical images and expressed as a percentage of total DAPI + cells ( n = 3). C , Pregnant female mice were subjected to a 24 h BrdU pulse before being killed. Ki67 (red) and BrdU (green) immunostaining was used to determine the percentage of cells exiting the cell cycle in control and Ctcf Nes-cre E14 cortex. Arrowheads indicate BrdU + Ki67 − cells that have exited the cell cycle. D , Cell cycle exit indices were calculated by measuring the ratio of BrdU + Ki67 − cells to total BrdU + cells in control and Ctcf Nes-cre cortex at E14 ( n = 3). E , Immunodetecti on of TBR1 (red) and SATB2 (green) in E14 control and Ctcf Nes-cre cortex. F , Immunodetection of CTIP2 (green) in E14 control and Ctcf Nes-cre cortex. G , SATB2 + , TBR1 + , and CTIP2 + cells were quantified and expressed as a percentage of DAPI + cells ( n = 3). Error bars represent SEM. Original magnification: A , C , E , F , 100×. Scale bars: A , C , 50 μm; E , F , 50 μm.
    Figure Legend Snippet: Ctcf -deficiency causes premature neurogenesis. A , Immunodetection of SOX2 (red) and TBR2 (green) in E14 control and Ctcf Nes-cre cortex. B , SOX2 + , TBR2 + , and SOX2 + /TBR2 + cells were quantified in 150-μm-wide cortical images and expressed as a percentage of total DAPI + cells ( n = 3). C , Pregnant female mice were subjected to a 24 h BrdU pulse before being killed. Ki67 (red) and BrdU (green) immunostaining was used to determine the percentage of cells exiting the cell cycle in control and Ctcf Nes-cre E14 cortex. Arrowheads indicate BrdU + Ki67 − cells that have exited the cell cycle. D , Cell cycle exit indices were calculated by measuring the ratio of BrdU + Ki67 − cells to total BrdU + cells in control and Ctcf Nes-cre cortex at E14 ( n = 3). E , Immunodetecti on of TBR1 (red) and SATB2 (green) in E14 control and Ctcf Nes-cre cortex. F , Immunodetection of CTIP2 (green) in E14 control and Ctcf Nes-cre cortex. G , SATB2 + , TBR1 + , and CTIP2 + cells were quantified and expressed as a percentage of DAPI + cells ( n = 3). Error bars represent SEM. Original magnification: A , C , E , F , 100×. Scale bars: A , C , 50 μm; E , F , 50 μm.

    Techniques Used: Immunodetection, Mouse Assay, Immunostaining

    Ctcf -deficiency results in PUMA-dependent apoptosis of apical and outer radial glia progenitors. Apical and outer radial glia progenitors that are rescued from apoptotic death fail to proliferate. Pregnant females were subjected to a 1 h BrdU pulse before being killed. A , SOX2 (red) and BrdU (green) coimmunostaining of E16.5 cortical cryosections. The inset demonstrates fewer SOX2 + cells in the Ctcf Nes-cre IZ than control or Ctcf Nes-cre ;Puma −/− . B , SOX2 + cells were quantified in 200-μm-wide cortical images ( n = 3). C , SOX2 + /BrdU + cells were quantified in 200-μm-wide cortical images ( n = 3). D , Immunodetection of SOX2 (green) and PAX6 (red) in the E16.5 cortical IZ demonstrates restoration of oRG progenitors in Ctcf Nes-cre ;Puma −/− cortex compared to Ctcf Nes-cre . Arrowheads indicate SOX2 + /PAX6 + oRG cells. Error bars represent SEM. Original magnification: A , D , 100×. Scale bars: A , 50 μm; D , 10 μm.
    Figure Legend Snippet: Ctcf -deficiency results in PUMA-dependent apoptosis of apical and outer radial glia progenitors. Apical and outer radial glia progenitors that are rescued from apoptotic death fail to proliferate. Pregnant females were subjected to a 1 h BrdU pulse before being killed. A , SOX2 (red) and BrdU (green) coimmunostaining of E16.5 cortical cryosections. The inset demonstrates fewer SOX2 + cells in the Ctcf Nes-cre IZ than control or Ctcf Nes-cre ;Puma −/− . B , SOX2 + cells were quantified in 200-μm-wide cortical images ( n = 3). C , SOX2 + /BrdU + cells were quantified in 200-μm-wide cortical images ( n = 3). D , Immunodetection of SOX2 (green) and PAX6 (red) in the E16.5 cortical IZ demonstrates restoration of oRG progenitors in Ctcf Nes-cre ;Puma −/− cortex compared to Ctcf Nes-cre . Arrowheads indicate SOX2 + /PAX6 + oRG cells. Error bars represent SEM. Original magnification: A , D , 100×. Scale bars: A , 50 μm; D , 10 μm.

    Techniques Used: Immunodetection

    8) Product Images from "MicroRNA-200a Regulates Grb2 and Suppresses Differentiation of Mouse Embryonic Stem Cells into Endoderm and Mesoderm"

    Article Title: MicroRNA-200a Regulates Grb2 and Suppresses Differentiation of Mouse Embryonic Stem Cells into Endoderm and Mesoderm

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068990

    Effects of miR-200a in ES cells and ES cell differentiation. (A) The expression of miR-200a, miR-200b and miR-429 in ES cell diferentiation. (B) Brightfield images and alkaline phosphatase staining of ES cells without LIF at 72 hours post-transfection with miRNA-200a. The scale bar represents 100 µm. (C) Relative levels of Oct4, Nanog, Sox2 and Rex1 mRNA in control or miR-200a-transfected ES cells. (D) Representative immunofluorescence images of control and miR-200a overexpression after 10 days of EB formation. Red, layer markers; blue, nuclei. The scale bar represents 100 µm. (E) Expression levels of genes associated with the differentiated state in EBs in response to miR-200a expression. All data are shown as the means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. ***, p
    Figure Legend Snippet: Effects of miR-200a in ES cells and ES cell differentiation. (A) The expression of miR-200a, miR-200b and miR-429 in ES cell diferentiation. (B) Brightfield images and alkaline phosphatase staining of ES cells without LIF at 72 hours post-transfection with miRNA-200a. The scale bar represents 100 µm. (C) Relative levels of Oct4, Nanog, Sox2 and Rex1 mRNA in control or miR-200a-transfected ES cells. (D) Representative immunofluorescence images of control and miR-200a overexpression after 10 days of EB formation. Red, layer markers; blue, nuclei. The scale bar represents 100 µm. (E) Expression levels of genes associated with the differentiated state in EBs in response to miR-200a expression. All data are shown as the means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. ***, p

    Techniques Used: Cell Differentiation, Expressing, Staining, Transfection, Immunofluorescence, Over Expression, Two Tailed Test

    9) Product Images from "Hypothalamic stem cells control aging speed partly through exosomal miRNAs"

    Article Title: Hypothalamic stem cells control aging speed partly through exosomal miRNAs

    Journal: Nature

    doi: 10.1038/nature23282

    Viral injection and additional information on TK/GCV model a , Lentiviruses of CMV-promoter driven GFP were injected into the hypothalamic third ventricle (3V) of C57BL/6 mice via pre-implanted cannula. At 1 week after injection, brain sections were made and examined for GFP immunostaining. Scale bar, 50 μm. b, c , AgRP-Cre mice and POMC-Cre mice received hypothalamic 3V injection of Hsv-TK lentiviruses followed by GCV treatment and were examined 3 months later for the numbers of these neurons in the ARC through Cre immunostaining. d , AgRP-Cre mice and POMC-Cre mice were injected with rAAV2-FLEX-rev-ChR2:tdTomato viruses or vehicle into the ARC, followed by injection of Sox2 promoter-driven Hsv-TK lentivirus (TK) or control lentivirus (Con) into the hypothalamic third ventricle. GCV was administrated into the third ventricle twice per week for 3 weeks. Subsequently these mice were subjected to optogenetic stimulation-induced feeding response as described in Method. Food intake prior to and post optogenetic stimulation were also measured. e , MWM training information for Figure 1f . Images represent 4 independent experiments ( a ). ** p
    Figure Legend Snippet: Viral injection and additional information on TK/GCV model a , Lentiviruses of CMV-promoter driven GFP were injected into the hypothalamic third ventricle (3V) of C57BL/6 mice via pre-implanted cannula. At 1 week after injection, brain sections were made and examined for GFP immunostaining. Scale bar, 50 μm. b, c , AgRP-Cre mice and POMC-Cre mice received hypothalamic 3V injection of Hsv-TK lentiviruses followed by GCV treatment and were examined 3 months later for the numbers of these neurons in the ARC through Cre immunostaining. d , AgRP-Cre mice and POMC-Cre mice were injected with rAAV2-FLEX-rev-ChR2:tdTomato viruses or vehicle into the ARC, followed by injection of Sox2 promoter-driven Hsv-TK lentivirus (TK) or control lentivirus (Con) into the hypothalamic third ventricle. GCV was administrated into the third ventricle twice per week for 3 weeks. Subsequently these mice were subjected to optogenetic stimulation-induced feeding response as described in Method. Food intake prior to and post optogenetic stimulation were also measured. e , MWM training information for Figure 1f . Images represent 4 independent experiments ( a ). ** p

    Techniques Used: Injection, Mouse Assay, Immunostaining

    Aging-associated hypothalamic NSC loss and its impact on aging speed a , b , Hypothalamic sections from male C57BL/6 mice at indicated months (M) of age were immunostained for Sox2 ( a, b ) with Bmi1 ( a ) or Nestin ( b ). c–f , Mid-aged C57BL/6 mice (~15-month-old) were injected in hypothalamic 3V with Sox2 promoter-directed Hsv-TK (TK) vs. control (Con) lentiviruses ( c ), followed by Ganciclovir (GCV) vs. vehicle treatment, and examined for cell ablation ( d, e ) and physiology ( f ) after 3 months post viral injection. d, e : Numbers of Sox2-postive cells and NeuN-positive neurons in hypothalamic 3V wall, arcuate nucleus (ARC) or MBH. MWM: Morris Water Maze (see ED Fig. 2e ). Images represent 3 independent experiments ( a–c ), scale bar, 100 μm. * p
    Figure Legend Snippet: Aging-associated hypothalamic NSC loss and its impact on aging speed a , b , Hypothalamic sections from male C57BL/6 mice at indicated months (M) of age were immunostained for Sox2 ( a, b ) with Bmi1 ( a ) or Nestin ( b ). c–f , Mid-aged C57BL/6 mice (~15-month-old) were injected in hypothalamic 3V with Sox2 promoter-directed Hsv-TK (TK) vs. control (Con) lentiviruses ( c ), followed by Ganciclovir (GCV) vs. vehicle treatment, and examined for cell ablation ( d, e ) and physiology ( f ) after 3 months post viral injection. d, e : Numbers of Sox2-postive cells and NeuN-positive neurons in hypothalamic 3V wall, arcuate nucleus (ARC) or MBH. MWM: Morris Water Maze (see ED Fig. 2e ). Images represent 3 independent experiments ( a–c ), scale bar, 100 μm. * p

    Techniques Used: Mouse Assay, Injection

    Ablation of htNSC in hypothalamic 3V wall by DTR/DT Mid-aged male C57BL/6 mice (~15-month-old) were injected in the hypothalamic 3V with Sox2 promoter-directed DTR lentivirus (DTR) or control lentivirus, followed by 4-week i.p. injection of DT or vehicle. a , Diagram of lentiviral DTR. b , Evaluation on Sox2 promoter-driven DTR lentiviruses in cultured htNSC via immunostaining for DTR and Sox2. Scale bar, 50 μm. c–e , Immunostaining of hypothalamic sections ( c, d ) and physiological analyses ( e ) of these mice at 3 months post viral injection. Control values in c and d represent similar observations in DTR-/DT+ and DTR+/DT- groups. Images represent 3 independent experiments ( b ). * p
    Figure Legend Snippet: Ablation of htNSC in hypothalamic 3V wall by DTR/DT Mid-aged male C57BL/6 mice (~15-month-old) were injected in the hypothalamic 3V with Sox2 promoter-directed DTR lentivirus (DTR) or control lentivirus, followed by 4-week i.p. injection of DT or vehicle. a , Diagram of lentiviral DTR. b , Evaluation on Sox2 promoter-driven DTR lentiviruses in cultured htNSC via immunostaining for DTR and Sox2. Scale bar, 50 μm. c–e , Immunostaining of hypothalamic sections ( c, d ) and physiological analyses ( e ) of these mice at 3 months post viral injection. Control values in c and d represent similar observations in DTR-/DT+ and DTR+/DT- groups. Images represent 3 independent experiments ( b ). * p

    Techniques Used: Mouse Assay, Injection, Cell Culture, Immunostaining

    Aging acceleration and lifespan shortening due to hypothalamic NSC loss Mid-aged male C57BL/6 mice (~15-month-old) were bilaterally injected in the MBH with Bmi1 promoter-driven Hsv-TK (TK) vs. control (Con) lentiviruses, followed by GCV vs. vehicle (Veh) treatment. a , Diagram of TK lentivirus and injection. b – d , Bmi1-positive cells in hypothalamic 3V wall and ARC ( b , c ) and NeuN-positive neurons ( d ) in the MBH at 3 months post viral injection. e–k , At 3~4 months post viral injection, TK+/GCV+ group (labeled as “TK/GCV”) were compared to control groups (data derived from TK+/GCV- group but also represented TK-/GCV+ and TK-/GCV- groups) for physiology ( e–j ) and histology ( k ). Scale bar, 100 μm. l – n , male C57BL/6 mice (8-month-old) were bilaterally injected in the MBH with Sox2 promoter-driven Hsv-TK (TK) vs. control (Con) lentiviruses, both followed by GCV treatment. l , Diagram of TK lentivirus and injection. m , Sox2-positive cells in MBH parenchyma and hypothalamic 3V wall of these mice (aged-matched un-injected mice included as a reference). n , Lifespan follow-up. Images represent 2 independent experiments ( k ) * p
    Figure Legend Snippet: Aging acceleration and lifespan shortening due to hypothalamic NSC loss Mid-aged male C57BL/6 mice (~15-month-old) were bilaterally injected in the MBH with Bmi1 promoter-driven Hsv-TK (TK) vs. control (Con) lentiviruses, followed by GCV vs. vehicle (Veh) treatment. a , Diagram of TK lentivirus and injection. b – d , Bmi1-positive cells in hypothalamic 3V wall and ARC ( b , c ) and NeuN-positive neurons ( d ) in the MBH at 3 months post viral injection. e–k , At 3~4 months post viral injection, TK+/GCV+ group (labeled as “TK/GCV”) were compared to control groups (data derived from TK+/GCV- group but also represented TK-/GCV+ and TK-/GCV- groups) for physiology ( e–j ) and histology ( k ). Scale bar, 100 μm. l – n , male C57BL/6 mice (8-month-old) were bilaterally injected in the MBH with Sox2 promoter-driven Hsv-TK (TK) vs. control (Con) lentiviruses, both followed by GCV treatment. l , Diagram of TK lentivirus and injection. m , Sox2-positive cells in MBH parenchyma and hypothalamic 3V wall of these mice (aged-matched un-injected mice included as a reference). n , Lifespan follow-up. Images represent 2 independent experiments ( k ) * p

    Techniques Used: Mouse Assay, Injection, Labeling, Derivative Assay

    Slowdown of aging by treatment of hypothalamic NSC-derived exosomes a , GFP-expressing htNSC transfected with Cy3-labeled miRNAs (106a-5p, 20a-5p and 466m-5p) were co-cultured with htNSC which did not contain GFP. Right panels: high-magnification images of representative GFP-positive and GFP-negative cells. Scale bar, 20 μm. b , Mid-aged C57BL/6 mice were treated via hypothalamic 3V cannula with hypothalamic NSC-derived exosomes vs. vehicle, 3 times per week for 4 months, and examined for Bmi1 and Sox2 in the MBH. Scale bar, 50 μm. c , Male C57BL/6 mice (15-month-old) were bilaterally injected in the MBH with Bmi1 promoter-driven Hsv-TK (TK) or control (Con) lentiviruses, followed by GCV vs. vehicle (Veh) treatment, treated in hypothalamic 3V with hypothalamic NSC-derived exosomes (2–3 times per week, 3 months), and examined for physiology. d , Male C57BL/6 mice (16-month-old) were treated in hypothalamic 3V with exosomes vs. Veh (3 times per week, 4 months) and examined for physiology ( d ). Images represent 3 independent experiments ( a, b ). * p
    Figure Legend Snippet: Slowdown of aging by treatment of hypothalamic NSC-derived exosomes a , GFP-expressing htNSC transfected with Cy3-labeled miRNAs (106a-5p, 20a-5p and 466m-5p) were co-cultured with htNSC which did not contain GFP. Right panels: high-magnification images of representative GFP-positive and GFP-negative cells. Scale bar, 20 μm. b , Mid-aged C57BL/6 mice were treated via hypothalamic 3V cannula with hypothalamic NSC-derived exosomes vs. vehicle, 3 times per week for 4 months, and examined for Bmi1 and Sox2 in the MBH. Scale bar, 50 μm. c , Male C57BL/6 mice (15-month-old) were bilaterally injected in the MBH with Bmi1 promoter-driven Hsv-TK (TK) or control (Con) lentiviruses, followed by GCV vs. vehicle (Veh) treatment, treated in hypothalamic 3V with hypothalamic NSC-derived exosomes (2–3 times per week, 3 months), and examined for physiology. d , Male C57BL/6 mice (16-month-old) were treated in hypothalamic 3V with exosomes vs. Veh (3 times per week, 4 months) and examined for physiology ( d ). Images represent 3 independent experiments ( a, b ). * p

    Techniques Used: Derivative Assay, Expressing, Transfection, Labeling, Cell Culture, Mouse Assay, Injection

    Contribution of hypothalamic NSC to exosomal miRNAs in the CSF a , Exosomal miRNAs in the same volume of CSF from young vs. mid-aged C57BL/6 mice (each miRNA in young group normalized as 1). b , Cultured hypothalamic NSC were co-infected with Cre-dependent Rab27a shRNA (shRNA-1 or -2) vs. control scramble shRNA lentiviruses with Sox2 promoter-driven Cre lentiviruses, and examined for Rab27a mRNA, secreted exosomes in the medium, and expression of indicated miRNAs in purified secreted exomsomes. c , C57BL/6 mice (4-month-old) were injected in hypothalamic 3V with Sox2 promoter-driven Cre lentiviruses and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentiviruses. CSF samples collected at 1 week post injection were analyzed for miRNAs. * p
    Figure Legend Snippet: Contribution of hypothalamic NSC to exosomal miRNAs in the CSF a , Exosomal miRNAs in the same volume of CSF from young vs. mid-aged C57BL/6 mice (each miRNA in young group normalized as 1). b , Cultured hypothalamic NSC were co-infected with Cre-dependent Rab27a shRNA (shRNA-1 or -2) vs. control scramble shRNA lentiviruses with Sox2 promoter-driven Cre lentiviruses, and examined for Rab27a mRNA, secreted exosomes in the medium, and expression of indicated miRNAs in purified secreted exomsomes. c , C57BL/6 mice (4-month-old) were injected in hypothalamic 3V with Sox2 promoter-driven Cre lentiviruses and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentiviruses. CSF samples collected at 1 week post injection were analyzed for miRNAs. * p

    Techniques Used: Mouse Assay, Cell Culture, Infection, shRNA, Expressing, Purification, Injection

    Effects on htNSC and animal physiology by Rab27a shRNA a, b , Cultured htNSC were infected with Sox2 promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentivirus ( a ) and examined for Ki67 via immunostaining ( b ) at 2–3 days post viral infection (Scale bars, 60 μm). c , C57BL/6 mice (12-month-old males) were injected in the hypothalamic 3V with Sox2 promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentivirus or vehicle. Aging-related physiology was analyzed in mice at 6 weeks post viral injection. * p
    Figure Legend Snippet: Effects on htNSC and animal physiology by Rab27a shRNA a, b , Cultured htNSC were infected with Sox2 promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentivirus ( a ) and examined for Ki67 via immunostaining ( b ) at 2–3 days post viral infection (Scale bars, 60 μm). c , C57BL/6 mice (12-month-old males) were injected in the hypothalamic 3V with Sox2 promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA vs. control scramble shRNA lentivirus or vehicle. Aging-related physiology was analyzed in mice at 6 weeks post viral injection. * p

    Techniques Used: shRNA, Cell Culture, Infection, Immunostaining, Mouse Assay, Injection

    10) Product Images from "Transient hypothyroidism favors oligodendrocyte generation providing functional remyelination in the adult mouse brain"

    Article Title: Transient hypothyroidism favors oligodendrocyte generation providing functional remyelination in the adult mouse brain

    Journal: eLife

    doi: 10.7554/eLife.29996

    Expression of the inactivating deiodinase, Dio3, excludes TH signalling from NSC, progenitors and oligodendrocyte progenitors. ( A ) The SVZ from nestin:GFP adult mice was double-stained with anti-GFAP (red) and anti-Dio3 (grey) antibodies. Note that Dio3 is highly expressed in the radial process of Nestin + and GFAP + NSC (arrowheads). ( B ) Correlations between the expression levels of Dio3 (black, y axis) and EGFR (red, x axis) versus DCX (blue, x axis) expression levels. ( C and D ) Coronal sections across the lateral ventricle of nestin:GFP adult mouse were stained with anti-SOX2 (red, C ), anti-DCX (blue, C and D ) and anti-Dio3 (grey, D ). Note that Dio3 shows much lower expression in DCX + neuroblasts but is highly expressed in Nestin (GFP + ) and SOX2 + progenitors. ( E ) Immunolabelling for EGFR (red), DCX (green) and Dio3 (grey) was performed on cells dissociated from neurospheres. Dio3 is strongly expressed in EGFR + progenitors and much lower expressed in DCX + neuroblasts.
    Figure Legend Snippet: Expression of the inactivating deiodinase, Dio3, excludes TH signalling from NSC, progenitors and oligodendrocyte progenitors. ( A ) The SVZ from nestin:GFP adult mice was double-stained with anti-GFAP (red) and anti-Dio3 (grey) antibodies. Note that Dio3 is highly expressed in the radial process of Nestin + and GFAP + NSC (arrowheads). ( B ) Correlations between the expression levels of Dio3 (black, y axis) and EGFR (red, x axis) versus DCX (blue, x axis) expression levels. ( C and D ) Coronal sections across the lateral ventricle of nestin:GFP adult mouse were stained with anti-SOX2 (red, C ), anti-DCX (blue, C and D ) and anti-Dio3 (grey, D ). Note that Dio3 shows much lower expression in DCX + neuroblasts but is highly expressed in Nestin (GFP + ) and SOX2 + progenitors. ( E ) Immunolabelling for EGFR (red), DCX (green) and Dio3 (grey) was performed on cells dissociated from neurospheres. Dio3 is strongly expressed in EGFR + progenitors and much lower expressed in DCX + neuroblasts.

    Techniques Used: Expressing, Mouse Assay, Staining

    Asymmetric TRα1/EGFR segregation in the adult SVZ. ( A–F ) TRα1 and EGFR segregate asymmetrically in the adult SVZ in vivo . Coronal sections of WT adult mouse brain across the lateral ventricles were immunostained with anti-TRα1 (red) and/or anti-EGFR (C-E’, green) or anti-SOX2 (F, green) antibodies. Mitotic figures in the SVZ were identified under confocal microscopy ( A–F ) and 3D reconstructions ( A’, B’, D’, E’ ) performed. ( A and A’ ) During prophase, TRα1 is preferentially localized at one pole of the dividing cell. ( B and B’ ) In telophase, only one of the daughters (left cell) has inherited largely TRα1. The other (on the right) shows residual TRα1 expression. ( C ) During telophase, only one daughter cell (lower cell) inherited EGFR. ( D and D’ ) Double labeling for TRα1 and EGFR shows opposite pole localizations during prophase. ( E and E’ ) During telophase, the left daughter is enriched in TRα1 whereas the cell on the right received predominately EGFR. ( F ) Asymmetric distribution of TRα1 (upper pole) occurs in SOX2 + progenitors Scale bar: 5 µm.
    Figure Legend Snippet: Asymmetric TRα1/EGFR segregation in the adult SVZ. ( A–F ) TRα1 and EGFR segregate asymmetrically in the adult SVZ in vivo . Coronal sections of WT adult mouse brain across the lateral ventricles were immunostained with anti-TRα1 (red) and/or anti-EGFR (C-E’, green) or anti-SOX2 (F, green) antibodies. Mitotic figures in the SVZ were identified under confocal microscopy ( A–F ) and 3D reconstructions ( A’, B’, D’, E’ ) performed. ( A and A’ ) During prophase, TRα1 is preferentially localized at one pole of the dividing cell. ( B and B’ ) In telophase, only one of the daughters (left cell) has inherited largely TRα1. The other (on the right) shows residual TRα1 expression. ( C ) During telophase, only one daughter cell (lower cell) inherited EGFR. ( D and D’ ) Double labeling for TRα1 and EGFR shows opposite pole localizations during prophase. ( E and E’ ) During telophase, the left daughter is enriched in TRα1 whereas the cell on the right received predominately EGFR. ( F ) Asymmetric distribution of TRα1 (upper pole) occurs in SOX2 + progenitors Scale bar: 5 µm.

    Techniques Used: In Vivo, Confocal Microscopy, Expressing, Labeling

    11) Product Images from "Generation and characterization of bat-induced pluripotent stem cells"

    Article Title: Generation and characterization of bat-induced pluripotent stem cells

    Journal: Theriogenology

    doi: 10.1016/j.theriogenology.2014.04.001

    Expression of pluripotency factors in biPSCs. (A) Real-time PCR analysis of the relative RNA expression levels of pluripotency genes (Oct4, Sox2, cMyc, and Klf4) in biPSCs versus that in BEFs. Data were normalized to β-actin mRNA expression levels and error bars were calculated by standard deviation (SD), n = 3. (B) The immunofluorescence staining of pluripotency markers Oct4, Sox2, Nanog, TBX3, and TRA-1-60 of biPSC colonies are shown (fluorescence, upper; 4,6-diamidino-2-phenylindole, middle; bright field, bottom). (C) Western blot analysis of Oct4 and Sox2 proteins. BiPSCs protein expression levels of BEFs and mouse ESCs were used as negative and positive controls, respectively. β-Actin was used as the loading control. biPSC, bat-induced pluripotent stem cell; PCR, polymerase chain reaction; BEF, bat embryonic fibroblast; ESC, embryonic stem cell.
    Figure Legend Snippet: Expression of pluripotency factors in biPSCs. (A) Real-time PCR analysis of the relative RNA expression levels of pluripotency genes (Oct4, Sox2, cMyc, and Klf4) in biPSCs versus that in BEFs. Data were normalized to β-actin mRNA expression levels and error bars were calculated by standard deviation (SD), n = 3. (B) The immunofluorescence staining of pluripotency markers Oct4, Sox2, Nanog, TBX3, and TRA-1-60 of biPSC colonies are shown (fluorescence, upper; 4,6-diamidino-2-phenylindole, middle; bright field, bottom). (C) Western blot analysis of Oct4 and Sox2 proteins. BiPSCs protein expression levels of BEFs and mouse ESCs were used as negative and positive controls, respectively. β-Actin was used as the loading control. biPSC, bat-induced pluripotent stem cell; PCR, polymerase chain reaction; BEF, bat embryonic fibroblast; ESC, embryonic stem cell.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Standard Deviation, Staining, Fluorescence, Western Blot, Polymerase Chain Reaction

    12) Product Images from "Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis"

    Article Title: Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2017.09.030

    Pax6 in late RPCs is required for generation of bipolar and glycinergic amacrine cells Double-immunolabeling with antibodies against GFP and cell-type-specific markers of electroporated retinas: syntaxin for amacrine interneurons (A,D), Vsx2 for bipolar interneurons (B,E), Sox2 for Müller glia (C,F), GABA for GABAergic amacrines (G,J), glycine transporter 1 (GlyT1) for glycinergic amacrines (H,K), Satb2 for nGnG amacrines (I,L). Arrowheads – co-localized cells. (M) Quantification of the number of marker-positive cells out of total GFP + cells in the inner nuclear layer (INL) reveals an increase in amacrine cells ( P = 0.045), a decrease in bipolar cells ( P = 0.008), and an increase in Müller glia ( P = 0.046); Stx, syntaxin. The glycinergic amacrine subclass is not detected in the mutant cells, whereas there was no significant change in the proportion of GABAergic ( P = 0.2) or nGnG ( P = 0.27) amacrines, N = 3 for all samples. Scale bar = 25 µm.
    Figure Legend Snippet: Pax6 in late RPCs is required for generation of bipolar and glycinergic amacrine cells Double-immunolabeling with antibodies against GFP and cell-type-specific markers of electroporated retinas: syntaxin for amacrine interneurons (A,D), Vsx2 for bipolar interneurons (B,E), Sox2 for Müller glia (C,F), GABA for GABAergic amacrines (G,J), glycine transporter 1 (GlyT1) for glycinergic amacrines (H,K), Satb2 for nGnG amacrines (I,L). Arrowheads – co-localized cells. (M) Quantification of the number of marker-positive cells out of total GFP + cells in the inner nuclear layer (INL) reveals an increase in amacrine cells ( P = 0.045), a decrease in bipolar cells ( P = 0.008), and an increase in Müller glia ( P = 0.046); Stx, syntaxin. The glycinergic amacrine subclass is not detected in the mutant cells, whereas there was no significant change in the proportion of GABAergic ( P = 0.2) or nGnG ( P = 0.27) amacrines, N = 3 for all samples. Scale bar = 25 µm.

    Techniques Used: Immunolabeling, Marker, Mutagenesis

    13) Product Images from "Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector"

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00415

    A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .
    Figure Legend Snippet: A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .

    Techniques Used: Injection, Expressing, Labeling, Marker

    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.
    Figure Legend Snippet: Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Techniques Used: Injection, Expressing, Labeling, Staining

    Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .
    Figure Legend Snippet: Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .

    Techniques Used: Labeling, In Vivo, In Vitro, Derivative Assay

    14) Product Images from "Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment"

    Article Title: Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00005

    Immunolabeling of FGF14 and selected neurogenesis markers in the DG using 1% formaldehyde and 0.5% MeOH fixation . (A) The gray and red channels represent FGF14 immunoreactivity visualized with an Alexa 568-conjugated secondary antibody, and the green channel represents Sox2, or DCX in (B) , visualized with an Alexa 488-conjugated secondary antibody. Green and red channels overlaid images are shown in the right column of both (A,B) . (C) The green channel represents Ankyrin-G (NeuroMab, catalog number 75-146) visualized with an Alexa 488-conjugated secondary antibody. The red channel represents BrdU immunoreactivity visualized with an Alexa 568-conjugated secondary antibody and the blue represents NeuN visualized with an Alexa 647-conjugated secondary antibody. DG, dentate gyrus; Sox2, Sex determining region Y-Box 2; DCX, doublecortin; BrdU, Bromodeoxyuridine; NeuN, Neuronal marker. Scale bars represent 20 μm.
    Figure Legend Snippet: Immunolabeling of FGF14 and selected neurogenesis markers in the DG using 1% formaldehyde and 0.5% MeOH fixation . (A) The gray and red channels represent FGF14 immunoreactivity visualized with an Alexa 568-conjugated secondary antibody, and the green channel represents Sox2, or DCX in (B) , visualized with an Alexa 488-conjugated secondary antibody. Green and red channels overlaid images are shown in the right column of both (A,B) . (C) The green channel represents Ankyrin-G (NeuroMab, catalog number 75-146) visualized with an Alexa 488-conjugated secondary antibody. The red channel represents BrdU immunoreactivity visualized with an Alexa 568-conjugated secondary antibody and the blue represents NeuN visualized with an Alexa 647-conjugated secondary antibody. DG, dentate gyrus; Sox2, Sex determining region Y-Box 2; DCX, doublecortin; BrdU, Bromodeoxyuridine; NeuN, Neuronal marker. Scale bars represent 20 μm.

    Techniques Used: Immunolabeling, Marker

    15) Product Images from "Harmine stimulates proliferation of human neural progenitors"

    Article Title: Harmine stimulates proliferation of human neural progenitors

    Journal: PeerJ

    doi: 10.7717/peerj.2727

    Characterization of neural progenitor cells derived from human embryonic stem cells. Representative images showing hNPCs stained for (A) TBR2 and Nestin, (B) PAX6 and β -Tubulin III, (C) GFAP and MAP2, (D) SOX2, (E) FOXG1, (F) DYRK1A. (G) Quantification of cell markers. (H) Venn diagram showing percentage of double stained cells. A minimum of 10,000 hNPCs was counted per marker. Scale bar: 25 µm.
    Figure Legend Snippet: Characterization of neural progenitor cells derived from human embryonic stem cells. Representative images showing hNPCs stained for (A) TBR2 and Nestin, (B) PAX6 and β -Tubulin III, (C) GFAP and MAP2, (D) SOX2, (E) FOXG1, (F) DYRK1A. (G) Quantification of cell markers. (H) Venn diagram showing percentage of double stained cells. A minimum of 10,000 hNPCs was counted per marker. Scale bar: 25 µm.

    Techniques Used: Derivative Assay, Staining, Marker

    16) Product Images from "Sox9 is critical for suppression of neurogenesis but not initiation of gliogenesis in the cerebellum"

    Article Title: Sox9 is critical for suppression of neurogenesis but not initiation of gliogenesis in the cerebellum

    Journal: Molecular Brain

    doi: 10.1186/s13041-015-0115-0

    Self-renewal of neural progenitors in cerebellum was not abrogated upon Sox9 inactivation as revealed by immunofluorescence staining on cerebellar sagittal sections. (A-D) Expression of the neural stem cell marker Sox2 was not lost in the Sox9- null cerebellum during late gestation under both En1-Cre and Pax2-Cre mediated Sox9 inactivation. In both groups, scattered signals were observed in the VZ and the prospective white matter, while the EGL displayed the strongest signal which corresponded to the actively proliferating granule cell precursors. (A’) and (B’) showed enlarged view of the VZ. (E-J) Quantification of mitotic activity at the VZ by double pulses of BrdU at either E16.5 (E-F) or E18.5 (G-H) did not reveal any difference in the proliferation of VZ neural progenitors between control and the Pax2-Cre induced Sox9 -null mutant. The number of BrdU-labeled cells in the VZ was divided by the total number of VZ cells to obtain the labeling index (I-J) . No statistically significant difference was observed using the Student’s t -test at both E16.5 (I; p = 0.4906) and E18.5 (J; p = 0.1633). (K-R) In addition, we did not observe any alterations in the expression of proliferation associated markers PCNA and Ki67 upon Sox9 inactivation. (A’-Q’) are the enlarged view at the VZ of (A-Q) . The error bars indicate standard deviations. Abbreviations: egl, external granular layer; pcl, Purkinje cell layer; rl, rhombic lip; vz, ventricular zone. Scale bars: A-S : 100 μm; A’-B’, G’-H’, K’-L’, P’-Q’: 50 μm.
    Figure Legend Snippet: Self-renewal of neural progenitors in cerebellum was not abrogated upon Sox9 inactivation as revealed by immunofluorescence staining on cerebellar sagittal sections. (A-D) Expression of the neural stem cell marker Sox2 was not lost in the Sox9- null cerebellum during late gestation under both En1-Cre and Pax2-Cre mediated Sox9 inactivation. In both groups, scattered signals were observed in the VZ and the prospective white matter, while the EGL displayed the strongest signal which corresponded to the actively proliferating granule cell precursors. (A’) and (B’) showed enlarged view of the VZ. (E-J) Quantification of mitotic activity at the VZ by double pulses of BrdU at either E16.5 (E-F) or E18.5 (G-H) did not reveal any difference in the proliferation of VZ neural progenitors between control and the Pax2-Cre induced Sox9 -null mutant. The number of BrdU-labeled cells in the VZ was divided by the total number of VZ cells to obtain the labeling index (I-J) . No statistically significant difference was observed using the Student’s t -test at both E16.5 (I; p = 0.4906) and E18.5 (J; p = 0.1633). (K-R) In addition, we did not observe any alterations in the expression of proliferation associated markers PCNA and Ki67 upon Sox9 inactivation. (A’-Q’) are the enlarged view at the VZ of (A-Q) . The error bars indicate standard deviations. Abbreviations: egl, external granular layer; pcl, Purkinje cell layer; rl, rhombic lip; vz, ventricular zone. Scale bars: A-S : 100 μm; A’-B’, G’-H’, K’-L’, P’-Q’: 50 μm.

    Techniques Used: Immunofluorescence, Staining, Expressing, Marker, Activity Assay, Mutagenesis, Labeling

    Sox9 expression coincides with markers for neural progenitors, RGCs and Bergmann glia during mouse cerebellum development as revealed by immunofluorescence staining of cerebellar sagittal sections from E11.5 to P5. (A, A’) At E11.5, Sox9 immunoreactivity could be observed in the entire cerebellar primordium, covering the newly specified VZ and RL. (B, B’) At E13.5, Sox9 expression became restricted to the VZ, while traces of Sox9+ cells were also found in the RL. (C, C’) Sox9 expression persisted throughout gestation at the VZ. From E15.5 onwards, Sox9 could also be detected in cells that had delaminated from the VZ as they migrate to the Purkinje cell plate at E18.5. (A’-C’) Magnified view of the corresponding VZ region marked by dotted square in A-C . Almost all the Sox9 expressing cells were immmunoreactive for the neural stem cell marker Sox2 throughout the embryonic cerebellum development. ( D and E ) Co-localization of Sox9 with the glial marker was observed from E13.5 to E18.5. (D’) At E13.5, EAAT1 was predominantly localized to the VZ where the Sox9+ radial glial cells reside. (E’) After the onset of astrogliogenesis, the majority of EAAT1 expression at E18.5 was found at the PCL where the Bergmann glial cells locate. ( F and F’ ) By P5, Sox9 signal was found predominantly at the PCL, where it labeled the soma of GFAP + Bergmann glial cells. Abbreviations: egl, external granular layer; igl, internal granular layer; pcl, Purkinje cell layer; ml, molecular layer; rl, rhombic lip; vz, ventricular zone. Scale bars: A-F, 100 μm; A’-F’, 50 μm.
    Figure Legend Snippet: Sox9 expression coincides with markers for neural progenitors, RGCs and Bergmann glia during mouse cerebellum development as revealed by immunofluorescence staining of cerebellar sagittal sections from E11.5 to P5. (A, A’) At E11.5, Sox9 immunoreactivity could be observed in the entire cerebellar primordium, covering the newly specified VZ and RL. (B, B’) At E13.5, Sox9 expression became restricted to the VZ, while traces of Sox9+ cells were also found in the RL. (C, C’) Sox9 expression persisted throughout gestation at the VZ. From E15.5 onwards, Sox9 could also be detected in cells that had delaminated from the VZ as they migrate to the Purkinje cell plate at E18.5. (A’-C’) Magnified view of the corresponding VZ region marked by dotted square in A-C . Almost all the Sox9 expressing cells were immmunoreactive for the neural stem cell marker Sox2 throughout the embryonic cerebellum development. ( D and E ) Co-localization of Sox9 with the glial marker was observed from E13.5 to E18.5. (D’) At E13.5, EAAT1 was predominantly localized to the VZ where the Sox9+ radial glial cells reside. (E’) After the onset of astrogliogenesis, the majority of EAAT1 expression at E18.5 was found at the PCL where the Bergmann glial cells locate. ( F and F’ ) By P5, Sox9 signal was found predominantly at the PCL, where it labeled the soma of GFAP + Bergmann glial cells. Abbreviations: egl, external granular layer; igl, internal granular layer; pcl, Purkinje cell layer; ml, molecular layer; rl, rhombic lip; vz, ventricular zone. Scale bars: A-F, 100 μm; A’-F’, 50 μm.

    Techniques Used: Expressing, Immunofluorescence, Staining, Marker, Labeling

    17) Product Images from "A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development"

    Article Title: A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.04.002

    Induction of NMPs Requires Low Levels of RA Signaling (A) Schematic of in vitro differentiation conditions and diagram illustrating the different cell fate choices of epiblast cells in the presence of bFGF/CHIR signaling and variable levels of RA. (B) Immunohistochemistry at D3 of differentiation indicates that Aldh1a2 −/− ESCs exposed to bFGF/CHIR downregulate Sox2 and express the mesodermal markers T/Bra and Tbx6. WT ESCs differentiated under the same conditions co-express T/Bra and Sox2. (C) Exposure of cells to increased levels of RA from D2 to D3 eliminates NMP induction and instead induces an NPC identity, evident by the expression of Sox2 in the absence of T/Bra and Tbx6. (D) qRT-PCR analysis of the expression of Sox2 , T/Bra , Tbx6 , Msgn1 , Sox1 , and Wnt3a at D3 in Aldh1a2 −/− cells and WT ESCs treated with bFGF/CHIR or bFGF/CHIR/RA (RA 10 or 100 nM). Mesodermal markers are induced in Aldh1a2 −/− cells, whereas the expression of Sox2 and Sox1 is abolished. By contrast, increasing RA concentrations induce neural fate identity, characterized by the upregulation of Sox2 and Sox1 , whereas the expression of the mesodermal genes, T/Bra , Msgn1 , and Tbx6 is absent. Expression of Wnt3a is significantly dowregulated under RA conditions. See also Figure S2 . (E) Schematic of differentiation conditions used for Cdx 1,2,4−/− cells. (F) Immunohistochemistry at D3 indicates the induction of cells that co-express T/Bra and Sox2. Also, Tbx6 expression is initially induced in the Cdx 1,2,4−/− cells. (G) Although continuing exposure to CHIR results in WT cells predominantly adopting Tbx6-expressing mesodermal identity, Cdx 1,2,4−/− cells acquire a Sox2-expressing NPC identity. (H) Inhibition of RA signaling (with 1 μM BMS) partially restores mesodermal differentiation, revealed by the upregulation of T/Bra in the Cdx 1,2,4−/− cells. (I) qRT-PCR analysis of mesodermal genes T/Bra and Tbx6 , RA signaling pathway components Cyp26a1 , Aldh1a2, and Wnt and Fgf signaling ligands Wnt3a and Fgf8 in Cdx 1,2,4−/− and WT cells at D3 (NMP conditions), D5 (CHIR conditions), or D5 CHIR conditions with RA inhibition (1 μM BMS) from D3 to D5. In the Cdx 1,2,4−/− cells the expression of T/Bra is induced at D3 but at lower levels, and the expression of Aldh1a2 is substantially increased compared with WT ESCs. At D5, expression of mesodermal markers T/Bra and Tbx6 , as well as Wnt3a and Fgf8 , is downregulated in Cdx 1,2,4−/− cells. RA inhibition in the presence of Fgf signaling results in partial restoration of T/Bra and Fgf8 in the Cdx 1,2,4−/− cells. qRT-PCR data were normalized against β-actin . Error bars indicate SD of three biological replicates. See also Figure S3 . (J) Quantitation of Bra + , Sox2 + , Tbx6 + , or Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D5 (CHIR or CHIR/BMS) of differentiation. Error bars indicate SD of four randomly selected independent fields. PNT, pre-neural tube; PS, primitive streak.
    Figure Legend Snippet: Induction of NMPs Requires Low Levels of RA Signaling (A) Schematic of in vitro differentiation conditions and diagram illustrating the different cell fate choices of epiblast cells in the presence of bFGF/CHIR signaling and variable levels of RA. (B) Immunohistochemistry at D3 of differentiation indicates that Aldh1a2 −/− ESCs exposed to bFGF/CHIR downregulate Sox2 and express the mesodermal markers T/Bra and Tbx6. WT ESCs differentiated under the same conditions co-express T/Bra and Sox2. (C) Exposure of cells to increased levels of RA from D2 to D3 eliminates NMP induction and instead induces an NPC identity, evident by the expression of Sox2 in the absence of T/Bra and Tbx6. (D) qRT-PCR analysis of the expression of Sox2 , T/Bra , Tbx6 , Msgn1 , Sox1 , and Wnt3a at D3 in Aldh1a2 −/− cells and WT ESCs treated with bFGF/CHIR or bFGF/CHIR/RA (RA 10 or 100 nM). Mesodermal markers are induced in Aldh1a2 −/− cells, whereas the expression of Sox2 and Sox1 is abolished. By contrast, increasing RA concentrations induce neural fate identity, characterized by the upregulation of Sox2 and Sox1 , whereas the expression of the mesodermal genes, T/Bra , Msgn1 , and Tbx6 is absent. Expression of Wnt3a is significantly dowregulated under RA conditions. See also Figure S2 . (E) Schematic of differentiation conditions used for Cdx 1,2,4−/− cells. (F) Immunohistochemistry at D3 indicates the induction of cells that co-express T/Bra and Sox2. Also, Tbx6 expression is initially induced in the Cdx 1,2,4−/− cells. (G) Although continuing exposure to CHIR results in WT cells predominantly adopting Tbx6-expressing mesodermal identity, Cdx 1,2,4−/− cells acquire a Sox2-expressing NPC identity. (H) Inhibition of RA signaling (with 1 μM BMS) partially restores mesodermal differentiation, revealed by the upregulation of T/Bra in the Cdx 1,2,4−/− cells. (I) qRT-PCR analysis of mesodermal genes T/Bra and Tbx6 , RA signaling pathway components Cyp26a1 , Aldh1a2, and Wnt and Fgf signaling ligands Wnt3a and Fgf8 in Cdx 1,2,4−/− and WT cells at D3 (NMP conditions), D5 (CHIR conditions), or D5 CHIR conditions with RA inhibition (1 μM BMS) from D3 to D5. In the Cdx 1,2,4−/− cells the expression of T/Bra is induced at D3 but at lower levels, and the expression of Aldh1a2 is substantially increased compared with WT ESCs. At D5, expression of mesodermal markers T/Bra and Tbx6 , as well as Wnt3a and Fgf8 , is downregulated in Cdx 1,2,4−/− cells. RA inhibition in the presence of Fgf signaling results in partial restoration of T/Bra and Fgf8 in the Cdx 1,2,4−/− cells. qRT-PCR data were normalized against β-actin . Error bars indicate SD of three biological replicates. See also Figure S3 . (J) Quantitation of Bra + , Sox2 + , Tbx6 + , or Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D5 (CHIR or CHIR/BMS) of differentiation. Error bars indicate SD of four randomly selected independent fields. PNT, pre-neural tube; PS, primitive streak.

    Techniques Used: In Vitro, Immunohistochemistry, Expressing, Quantitative RT-PCR, Inhibition, Quantitation Assay

    NMP Cells Are Generated in the Absence of Msgn1 but Cannot Efficiently Differentiate to PSM Identity (A) Schematic of in vitro culture conditions used for assaying Msgn1 −/− cells. (B) Immunohistochemistry of Msgn1 −/− cells at D3 of differentiation reveals most cells co-express T/Bra and Sox2. The expression of Tbx6 is also evident in some cells at D3. (C) At D5 under mesodermal conditions WT ESCs predominantly differentiate to PSM, whereas Msgn1 −/− cells are maintained in an NMP state co-expressing T/Bra with Sox2. Tbx6 is also expressed in some cells, but these have not downregulated T/Bra. (D) Downregulation of T/Bra is delayed in the Msgn1 −/− cells under NB conditions, as is evident by the presence of T/Bra-expressing cells at D5. See also Figure S4 . (E) Quantitation of T/Bra + , Sox2 + , Tbx6 + , or T/Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D5 (CHIR) of Msgn1 −/− ESC differentiation. Error bars indicate SD of four randomly selected independent fields. (F) qRT-PCR analysis of T/Bra , Tbx6 , Cdx2 , Sox2 and Nkx1.2 at D3 (NMP conditions), D5-CHIR (mesodermal conditions) and D5 NB (neural conditions) in Msgn1 −/− and WT cells. At D3 Msgn1 −/− cells express high levels of T/Bra and lower levels of Tbx6 compared with controls. Cdx2 and Nkx1.2 expression is not affected, whereas Sox2 is downregulated. At D5 CHIR conditions Msgn1 −/− cells express high levels of NMP markers, T/Bra , Sox2 , Cdx2 , Nkx1.2 and lower levels of Tbx6 , compared with WT cells that have acquired a PSM identity. (G) The expression of Wnt3a and Fgf8 is significantly higher in the Msgn1 −/− cells at D5 CHIR, whereas the expression of Aldh1a2 is downregulated. qRT-PCR data were normalized relative to β-actin . Error bars indicate SD of three biological replicates.
    Figure Legend Snippet: NMP Cells Are Generated in the Absence of Msgn1 but Cannot Efficiently Differentiate to PSM Identity (A) Schematic of in vitro culture conditions used for assaying Msgn1 −/− cells. (B) Immunohistochemistry of Msgn1 −/− cells at D3 of differentiation reveals most cells co-express T/Bra and Sox2. The expression of Tbx6 is also evident in some cells at D3. (C) At D5 under mesodermal conditions WT ESCs predominantly differentiate to PSM, whereas Msgn1 −/− cells are maintained in an NMP state co-expressing T/Bra with Sox2. Tbx6 is also expressed in some cells, but these have not downregulated T/Bra. (D) Downregulation of T/Bra is delayed in the Msgn1 −/− cells under NB conditions, as is evident by the presence of T/Bra-expressing cells at D5. See also Figure S4 . (E) Quantitation of T/Bra + , Sox2 + , Tbx6 + , or T/Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D5 (CHIR) of Msgn1 −/− ESC differentiation. Error bars indicate SD of four randomly selected independent fields. (F) qRT-PCR analysis of T/Bra , Tbx6 , Cdx2 , Sox2 and Nkx1.2 at D3 (NMP conditions), D5-CHIR (mesodermal conditions) and D5 NB (neural conditions) in Msgn1 −/− and WT cells. At D3 Msgn1 −/− cells express high levels of T/Bra and lower levels of Tbx6 compared with controls. Cdx2 and Nkx1.2 expression is not affected, whereas Sox2 is downregulated. At D5 CHIR conditions Msgn1 −/− cells express high levels of NMP markers, T/Bra , Sox2 , Cdx2 , Nkx1.2 and lower levels of Tbx6 , compared with WT cells that have acquired a PSM identity. (G) The expression of Wnt3a and Fgf8 is significantly higher in the Msgn1 −/− cells at D5 CHIR, whereas the expression of Aldh1a2 is downregulated. qRT-PCR data were normalized relative to β-actin . Error bars indicate SD of three biological replicates.

    Techniques Used: Generated, In Vitro, Immunohistochemistry, Expressing, Quantitation Assay, Quantitative RT-PCR

    Reverse Engineering the NMP Gene Regulatory Network (A) Six dynamical patterns summarize the experimental observations used as objectives to identify the best-fit network topology. Targeted time points are squares positioned at observed gene levels for Sox2 (orange), T/Bra (blue), and Msgn1/Tbx6 (red). The x axis represents the simulated time between D2 and D5. The y axis represents the protein concentration in a.u. All simulations are performed with a deterministic ordinary differential equation model from the initial conditions: Sox2 HIGH , T/ Bra LOW , and Msgn1/Tbx6 LOW . In addition to WT simulations, T/Bra and Msgn1/Tbx6 mutant simulations were performed by setting the relevant protein production rate to zero. Dashed lines represents the four RA / Wnt signaling conditions and solid lines a typical best-fit solution. (B) Overview of the 64 parallel parameter explorations evaluated. Each bar plot represents the average score of the best-fit solution cluster for one topology (a perfect score is 0). Bar colors identify the six objectives shown in (A). Underneath, circles describe the associated topology (blue circles for repression, red circles for activation) and the radius of each circle is proportional to the binding affinities in the best-fit solutions. (C) The best-fit topology identified by the parameter exploration. (D) Results of stochastic simulations. Colored pie charts show the ratios of cell states observed at D5 of stochastic simulations using the optimal topology under various RA / Wnt conditions between D3 and D5. Prior to D3, all trajectories are simulated with the same RA LOW / Wnt HIGH signaling condition. (E) Stochastic trajectories obtained with four RA / Wnt signaling conditions that are representative of the four conditions in (D). The thick solid lines indicate the average trajectories for neural (green) and mesodermal (red) fates. Circles represent the attractors of the dynamical system. (F) Experimental results. The ratios of different cell types obtained by co-measuring Sox2, T/Bra, and Tbx6 levels by flow cytometry. Assays were conducted using six RA / Wnt signaling conditions between D3 and D4. Grayscale panels show 2D kernel density estimations for each cell obtained from the protein fluorophore intensities. Contour plots document the highest density regions of the four cell populations identified by k-means clustering. Axes follow the “logicle” scale ( Parks et al., 2006 ). Associated bar plots indicate the ratios of cell types in each condition.
    Figure Legend Snippet: Reverse Engineering the NMP Gene Regulatory Network (A) Six dynamical patterns summarize the experimental observations used as objectives to identify the best-fit network topology. Targeted time points are squares positioned at observed gene levels for Sox2 (orange), T/Bra (blue), and Msgn1/Tbx6 (red). The x axis represents the simulated time between D2 and D5. The y axis represents the protein concentration in a.u. All simulations are performed with a deterministic ordinary differential equation model from the initial conditions: Sox2 HIGH , T/ Bra LOW , and Msgn1/Tbx6 LOW . In addition to WT simulations, T/Bra and Msgn1/Tbx6 mutant simulations were performed by setting the relevant protein production rate to zero. Dashed lines represents the four RA / Wnt signaling conditions and solid lines a typical best-fit solution. (B) Overview of the 64 parallel parameter explorations evaluated. Each bar plot represents the average score of the best-fit solution cluster for one topology (a perfect score is 0). Bar colors identify the six objectives shown in (A). Underneath, circles describe the associated topology (blue circles for repression, red circles for activation) and the radius of each circle is proportional to the binding affinities in the best-fit solutions. (C) The best-fit topology identified by the parameter exploration. (D) Results of stochastic simulations. Colored pie charts show the ratios of cell states observed at D5 of stochastic simulations using the optimal topology under various RA / Wnt conditions between D3 and D5. Prior to D3, all trajectories are simulated with the same RA LOW / Wnt HIGH signaling condition. (E) Stochastic trajectories obtained with four RA / Wnt signaling conditions that are representative of the four conditions in (D). The thick solid lines indicate the average trajectories for neural (green) and mesodermal (red) fates. Circles represent the attractors of the dynamical system. (F) Experimental results. The ratios of different cell types obtained by co-measuring Sox2, T/Bra, and Tbx6 levels by flow cytometry. Assays were conducted using six RA / Wnt signaling conditions between D3 and D4. Grayscale panels show 2D kernel density estimations for each cell obtained from the protein fluorophore intensities. Contour plots document the highest density regions of the four cell populations identified by k-means clustering. Axes follow the “logicle” scale ( Parks et al., 2006 ). Associated bar plots indicate the ratios of cell types in each condition.

    Techniques Used: Protein Concentration, Mutagenesis, Activation Assay, Binding Assay, Flow Cytometry, Cytometry

    Single-Cell Analysis of the Differentiation Route of NMPs toward the Neural Lineage (A) Schematic of the differentiation conditions used for the generation of in vitro NMPs and neural progenitor cells (NPCs) from mESCs. (B) Immunohistochemistry of cultures at D3 indicates that most cells co-express Brachyury (T/Bra) and Sox2, characteristic of NMPs, and a small percentage of cells expressed Tbx6. Removal of CHIR after D3 and culture until D4 in NB (neurobasal) conditions induces the generation of NPCs that express Sox2 in the absence of T/Bra, and a few mesodermal cells expressing Tbx6. (C) Quantitation of T/Bra + , Sox2 + , Tbx6 + , or T/Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D4 (NB) of differentiation. Error bars indicate SD of four randomly selected independent fields. (D) Pseudotemporal ordering of D3 and D4 cells identifies four different populations and two distinct differentiation trajectories that lead to either neural or mesodermal identity. Expression of Cdx1 , Cdx2 , Cdx4 , T/Bra , and Nkx1.2 is high in NMP cells, the expression of mesoderm specific genes Msgn1 and Tbx6 is high in Mesoderm cells. By contrast, there is induction of Sox1 , Irx3 , and Zic2 along the neural trajectory. The expression of RA signaling pathway components is differentially regulated in each developmental trajectory. RXRγ and RARγ are co-expressed in D3 NMP cells (similar to e8.5 NMPs). Expression of Aldh1a2 correlated strongly with Msgn1 and Tbx6 expression. See also Figure S1 . (E) Hierarchical clustering of D3 and D4 single cells partitions the cells into four major groups. An NMP cluster (NMP, cerulean), a transitioning NMP (t-NMP, cyan) that express markers of developmentally older NMPs, a mesodermal (Meso, red), and a neural progenitor cell cluster (NPC, green). D3 cells are indicated in gray and D4 cells in black. See Tables S3 and S4 . (F) Diagram illustrating the mesoderm or neural progenitor fate choice made by NMP cells. (G) Schematic of the posterior part of an e8.5 mouse embryo. NMP cells (cerulean) expressing Bra/Sox2/Cdx genes are located in the CLE region, close to the NSB in the anterior part of the primitive streak. As cells leave the NMP zone they differentiate to MPC progenitors (cerulean/red) expressing Bra/Msgn1/Tbx6 , which results in upregulation of Aldh1a2 . Thus, increased levels of RA produced in close proximity to the niche promote Sox2 expression and the differentiation of NMPs to PNT cells (green/yellow) then NPCs (green). The transcriptional network that controls the cell fate decision of NMP cells toward neural or mesodermal identities is summarized adjacent to the embryo model. NMP, neuromesodermal progenitor; t-NMP, transitioning neuromesodermal progenitor; NPCs, neural progenitor cells; PSM, presomitic mesoderm; MPC, mesodermal progenitor cells; PNT, pre-neural tube cells; NSB, node-streak border; PS, primitive streak; NB, neurobasal conditions.
    Figure Legend Snippet: Single-Cell Analysis of the Differentiation Route of NMPs toward the Neural Lineage (A) Schematic of the differentiation conditions used for the generation of in vitro NMPs and neural progenitor cells (NPCs) from mESCs. (B) Immunohistochemistry of cultures at D3 indicates that most cells co-express Brachyury (T/Bra) and Sox2, characteristic of NMPs, and a small percentage of cells expressed Tbx6. Removal of CHIR after D3 and culture until D4 in NB (neurobasal) conditions induces the generation of NPCs that express Sox2 in the absence of T/Bra, and a few mesodermal cells expressing Tbx6. (C) Quantitation of T/Bra + , Sox2 + , Tbx6 + , or T/Bra + /Sox2 + signal + area normalized to DAPI area at D3 (bFGF/CHIR) and D4 (NB) of differentiation. Error bars indicate SD of four randomly selected independent fields. (D) Pseudotemporal ordering of D3 and D4 cells identifies four different populations and two distinct differentiation trajectories that lead to either neural or mesodermal identity. Expression of Cdx1 , Cdx2 , Cdx4 , T/Bra , and Nkx1.2 is high in NMP cells, the expression of mesoderm specific genes Msgn1 and Tbx6 is high in Mesoderm cells. By contrast, there is induction of Sox1 , Irx3 , and Zic2 along the neural trajectory. The expression of RA signaling pathway components is differentially regulated in each developmental trajectory. RXRγ and RARγ are co-expressed in D3 NMP cells (similar to e8.5 NMPs). Expression of Aldh1a2 correlated strongly with Msgn1 and Tbx6 expression. See also Figure S1 . (E) Hierarchical clustering of D3 and D4 single cells partitions the cells into four major groups. An NMP cluster (NMP, cerulean), a transitioning NMP (t-NMP, cyan) that express markers of developmentally older NMPs, a mesodermal (Meso, red), and a neural progenitor cell cluster (NPC, green). D3 cells are indicated in gray and D4 cells in black. See Tables S3 and S4 . (F) Diagram illustrating the mesoderm or neural progenitor fate choice made by NMP cells. (G) Schematic of the posterior part of an e8.5 mouse embryo. NMP cells (cerulean) expressing Bra/Sox2/Cdx genes are located in the CLE region, close to the NSB in the anterior part of the primitive streak. As cells leave the NMP zone they differentiate to MPC progenitors (cerulean/red) expressing Bra/Msgn1/Tbx6 , which results in upregulation of Aldh1a2 . Thus, increased levels of RA produced in close proximity to the niche promote Sox2 expression and the differentiation of NMPs to PNT cells (green/yellow) then NPCs (green). The transcriptional network that controls the cell fate decision of NMP cells toward neural or mesodermal identities is summarized adjacent to the embryo model. NMP, neuromesodermal progenitor; t-NMP, transitioning neuromesodermal progenitor; NPCs, neural progenitor cells; PSM, presomitic mesoderm; MPC, mesodermal progenitor cells; PNT, pre-neural tube cells; NSB, node-streak border; PS, primitive streak; NB, neurobasal conditions.

    Techniques Used: Single-cell Analysis, In Vitro, Immunohistochemistry, Expressing, Quantitation Assay, Produced

    Single-Cell Transcriptome Analysis of In Vivo NMPs Defines the Molecular Signature of e8.5 and e9.5 NMPs (A) Strategy for single-cell transcriptional analysis of e-NMPs dissected from the CLE region of e8.5 and e9.5 mouse embryos. (B) Hierarchical clustering of embryo-derived single cells, using the genes in the first three modules ( Table S1 ), columns represent cells and rows correspond to genes. This separates cells into two large groups correlating with developmental age (e8.5 and e9.5). Within the e8.5 group, three smaller clusters could be distinguished, an e8.5 NMP identity (expressing genes in module 1), MPCs (expressing genes in modules 1 and 3) and mesodermal cells (Meso) (expressing genes in module 3). The e9.5 group was subdivided into two groups, one associated with e9.5 NMP identity (module 2) the other with MPC fate (modules 2 and 3). The cluster identity of each cell from the e8.5 and e9.5 embryos is indicated in orange (e8.5 NMPs), purple (e9.5 NMPs), brown and pink (MPCs), and red (Meso). (C) Pseudotemporal ordering of cells (right) obtained via the associated cell state graph (left) (expression levels are indicated as normalized counts per million reads). The white-to-red colors indicate NMP-to-mesodermal fractional identity of each cells defined by Sox2 , Nkx1.2 , Msgn1 , Tbx6 , and Meox1 levels. (D) Developmental trajectories of single cells from e8.5 and e9.5 mouse embryos reveals three distinct populations, NMP (T/Bra + /Sox2 + ), MPC (T/Bra + /Msgn1 + /Tbx6 + ), and PSM (T/Bra − /Msgn1 + /Tbx6 + ). (E) Molecular signature of e-NMP cells identified by differential expression analysis of the e8.5 (top) and e9.5 (bottom) NMPs with PSM cells. (F) The analysis identified 68 genes that were associated with both e8.5 and e9.5 NMP identity and 31 genes associated with mesodermal differentiation. For clarity we have shown only the 31 genes most enriched in e-NMPs. The complete gene lists are given in Table S2 . (G) Differential expression analysis of e8.5 NMPs and e9.5 NMPs defines the molecular signature of e-NMPs at different developmental stages. Log CPM, logarithmic counts per million; Log f.c., logarithmic fold change; NMPs, neuromesodermal progenitors; MPC, mesodermal progenitors; PSM, presomitic mesoderm; SB, somite border.
    Figure Legend Snippet: Single-Cell Transcriptome Analysis of In Vivo NMPs Defines the Molecular Signature of e8.5 and e9.5 NMPs (A) Strategy for single-cell transcriptional analysis of e-NMPs dissected from the CLE region of e8.5 and e9.5 mouse embryos. (B) Hierarchical clustering of embryo-derived single cells, using the genes in the first three modules ( Table S1 ), columns represent cells and rows correspond to genes. This separates cells into two large groups correlating with developmental age (e8.5 and e9.5). Within the e8.5 group, three smaller clusters could be distinguished, an e8.5 NMP identity (expressing genes in module 1), MPCs (expressing genes in modules 1 and 3) and mesodermal cells (Meso) (expressing genes in module 3). The e9.5 group was subdivided into two groups, one associated with e9.5 NMP identity (module 2) the other with MPC fate (modules 2 and 3). The cluster identity of each cell from the e8.5 and e9.5 embryos is indicated in orange (e8.5 NMPs), purple (e9.5 NMPs), brown and pink (MPCs), and red (Meso). (C) Pseudotemporal ordering of cells (right) obtained via the associated cell state graph (left) (expression levels are indicated as normalized counts per million reads). The white-to-red colors indicate NMP-to-mesodermal fractional identity of each cells defined by Sox2 , Nkx1.2 , Msgn1 , Tbx6 , and Meox1 levels. (D) Developmental trajectories of single cells from e8.5 and e9.5 mouse embryos reveals three distinct populations, NMP (T/Bra + /Sox2 + ), MPC (T/Bra + /Msgn1 + /Tbx6 + ), and PSM (T/Bra − /Msgn1 + /Tbx6 + ). (E) Molecular signature of e-NMP cells identified by differential expression analysis of the e8.5 (top) and e9.5 (bottom) NMPs with PSM cells. (F) The analysis identified 68 genes that were associated with both e8.5 and e9.5 NMP identity and 31 genes associated with mesodermal differentiation. For clarity we have shown only the 31 genes most enriched in e-NMPs. The complete gene lists are given in Table S2 . (G) Differential expression analysis of e8.5 NMPs and e9.5 NMPs defines the molecular signature of e-NMPs at different developmental stages. Log CPM, logarithmic counts per million; Log f.c., logarithmic fold change; NMPs, neuromesodermal progenitors; MPC, mesodermal progenitors; PSM, presomitic mesoderm; SB, somite border.

    Techniques Used: In Vivo, Derivative Assay, Expressing

    Maintenance of NMP Cell Identity in the Absence of Tbx6 (A) Schematic of conditions to assay Tbx6 −/− ESCs. (B) Tbx6 −/− cells co-expressing T/Bra and Sox2 at D3 under NMP conditions (bFGF/CHIR). (C) Tbx6 −/− cells are maintained as NMPs characterized by the co-expression of T/Bra and Sox2 at D5 (CHIR conditions), whereas WT cells mostly downregulate T/Bra and Sox2 and instead express Tbx6. (D) At D5 in CHIR conditions, Tbx6 −/− cells acquire an identity more similar to e9.5 NMPs characterized by the co-expression of T/Bra + /Sox2 + with Hoxc10 + . In WT cells, few late NMP cells could be detected, as most Hoxc10-expressing cells were T/Bra negative. (E) qRT-PCR analysis of NMP, mesodermal, and neural markers at D3 (NMP conditions), D5 CHIR (mesodermal conditions), and D5 NB (neural conditions) shows that NMPs are induced at D3 in the Tbx6 −/− cells and maintained during exposure to CHIR. Expression of Aldh1a2 is upregulated at D3 and D5 (CHIR) in WT ESCs, whereas Tbx6 −/− cells express low levels of Aldh1a2 and higher levels of Fgf8 and Wnt3a at D5 (CHIR). (F and G) The Tbx6 −/− cells maintained NMP identity for 9 days (two passages) under bFGF/CHIR conditions (F) and progressively express more posterior Hox genes (G). (H) tSNE projection of the in vitro WT and Tbx6 −/− passaged NMPs with e8.5 and e9.5 NMPs revealed that D3 WT and Tbx6 −/− in vitro NMPs are similar to e8.5 NMPs, whereas the passaged D6 Tbx6 −/− in vitro NMPs closely resemble e9.5 NMPs. (I and J) Taking the intersection of differentially expressed genes identified between in vitro D3 Tbx6 −/− and D6 Tbx6 −/− cells, and in vivo e8.5 and e9.5 NMPs showed similar changes in gene expression of early (I) and late NMP signature genes (J).
    Figure Legend Snippet: Maintenance of NMP Cell Identity in the Absence of Tbx6 (A) Schematic of conditions to assay Tbx6 −/− ESCs. (B) Tbx6 −/− cells co-expressing T/Bra and Sox2 at D3 under NMP conditions (bFGF/CHIR). (C) Tbx6 −/− cells are maintained as NMPs characterized by the co-expression of T/Bra and Sox2 at D5 (CHIR conditions), whereas WT cells mostly downregulate T/Bra and Sox2 and instead express Tbx6. (D) At D5 in CHIR conditions, Tbx6 −/− cells acquire an identity more similar to e9.5 NMPs characterized by the co-expression of T/Bra + /Sox2 + with Hoxc10 + . In WT cells, few late NMP cells could be detected, as most Hoxc10-expressing cells were T/Bra negative. (E) qRT-PCR analysis of NMP, mesodermal, and neural markers at D3 (NMP conditions), D5 CHIR (mesodermal conditions), and D5 NB (neural conditions) shows that NMPs are induced at D3 in the Tbx6 −/− cells and maintained during exposure to CHIR. Expression of Aldh1a2 is upregulated at D3 and D5 (CHIR) in WT ESCs, whereas Tbx6 −/− cells express low levels of Aldh1a2 and higher levels of Fgf8 and Wnt3a at D5 (CHIR). (F and G) The Tbx6 −/− cells maintained NMP identity for 9 days (two passages) under bFGF/CHIR conditions (F) and progressively express more posterior Hox genes (G). (H) tSNE projection of the in vitro WT and Tbx6 −/− passaged NMPs with e8.5 and e9.5 NMPs revealed that D3 WT and Tbx6 −/− in vitro NMPs are similar to e8.5 NMPs, whereas the passaged D6 Tbx6 −/− in vitro NMPs closely resemble e9.5 NMPs. (I and J) Taking the intersection of differentially expressed genes identified between in vitro D3 Tbx6 −/− and D6 Tbx6 −/− cells, and in vivo e8.5 and e9.5 NMPs showed similar changes in gene expression of early (I) and late NMP signature genes (J).

    Techniques Used: Expressing, Quantitative RT-PCR, In Vitro, In Vivo

    18) Product Images from "Sub-Nanomolar Methylmercury Exposure Promotes Premature Differentiation of Murine Embryonic Neural Precursor at the Expense of Their Proliferation"

    Article Title: Sub-Nanomolar Methylmercury Exposure Promotes Premature Differentiation of Murine Embryonic Neural Precursor at the Expense of Their Proliferation

    Journal: Toxics

    doi: 10.3390/toxics6040061

    Immunocytochemistry was performed in a 3-day NSC culture. Images of ( A ) Pax6 (Green), Beta III Tubulin (Red), ( D ) Sox2 (Green), Ki67 (Red), and Hoechst (Blue) staining are shown. Scale bar = 50 μm. The bar graphs ( B , C ; E , F ) show the percentage of immunocytochemistry-positive cells. Values are mean ± SEM ( n = 3). Statistical significance was determined by a one-way ANOVA followed by a Bonferroni’s post hoc test (* p
    Figure Legend Snippet: Immunocytochemistry was performed in a 3-day NSC culture. Images of ( A ) Pax6 (Green), Beta III Tubulin (Red), ( D ) Sox2 (Green), Ki67 (Red), and Hoechst (Blue) staining are shown. Scale bar = 50 μm. The bar graphs ( B , C ; E , F ) show the percentage of immunocytochemistry-positive cells. Values are mean ± SEM ( n = 3). Statistical significance was determined by a one-way ANOVA followed by a Bonferroni’s post hoc test (* p

    Techniques Used: Immunocytochemistry, Staining

    19) Product Images from "Sox2 Up-regulation and Glial Cell Proliferation Following Degeneration of Spiral Ganglion Neurons in the Adult Mouse Inner Ear"

    Article Title: Sox2 Up-regulation and Glial Cell Proliferation Following Degeneration of Spiral Ganglion Neurons in the Adult Mouse Inner Ear

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    doi: 10.1007/s10162-010-0244-1

    Increased Sox2 + cells in the osseous spiral lamina in injured auditory nerves. A – C Glial cells within osseous spiral lamina of control ears are not stained with Sox2 antibody. Nuclei were counterstained with PI ( red ). D – F Many cells stained positively with Sox2 antibody ( green ) in a mouse 7 days after ouabain treatment. G Mean density of Sox2 + cells within the osseous spiral lamina in the apical, middle, and basal turns from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). Scale bar , 5 μm in F (applies to A – F ).
    Figure Legend Snippet: Increased Sox2 + cells in the osseous spiral lamina in injured auditory nerves. A – C Glial cells within osseous spiral lamina of control ears are not stained with Sox2 antibody. Nuclei were counterstained with PI ( red ). D – F Many cells stained positively with Sox2 antibody ( green ) in a mouse 7 days after ouabain treatment. G Mean density of Sox2 + cells within the osseous spiral lamina in the apical, middle, and basal turns from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). Scale bar , 5 μm in F (applies to A – F ).

    Techniques Used: Staining

    Increased BrdU + and BrdU + /Sox2 + cells in injured auditory nerve. A Dual immunostaining for Sox2 ( green ) and BrdU ( red , arrows ) in a normal auditory nerve showing only one BrdU + cell that was not co-labeled with Sox2 ( arrowheads ). B , C Increased number of BrdU + cells in mice 3 and 7 days after ouabain treatment. A majority of BrdU + cells were co-labeled with Sox2 ( yellow , arrows ). D Three-dimensional reconstructed confocal image shows a cell co-labeled for Sox2 and BrdU in a mouse 7 days after ouabain treatment. E Mean density of BrdU + cells within the Rosenthal’s canal in the apical, middle, and basal turns of control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). F Mean density of BrdU + /Sox2 + cells within Rosenthal’s canal. G Mean density of BrdU + /Sox2 + cells among the total BrdU + cells. H Mean density of BrdU + /Sox2 + cells among the total Sox2 + cells. Scale bars , 15 μm in C (applies to A – C ); 15 μm in D .
    Figure Legend Snippet: Increased BrdU + and BrdU + /Sox2 + cells in injured auditory nerve. A Dual immunostaining for Sox2 ( green ) and BrdU ( red , arrows ) in a normal auditory nerve showing only one BrdU + cell that was not co-labeled with Sox2 ( arrowheads ). B , C Increased number of BrdU + cells in mice 3 and 7 days after ouabain treatment. A majority of BrdU + cells were co-labeled with Sox2 ( yellow , arrows ). D Three-dimensional reconstructed confocal image shows a cell co-labeled for Sox2 and BrdU in a mouse 7 days after ouabain treatment. E Mean density of BrdU + cells within the Rosenthal’s canal in the apical, middle, and basal turns of control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). F Mean density of BrdU + /Sox2 + cells within Rosenthal’s canal. G Mean density of BrdU + /Sox2 + cells among the total BrdU + cells. H Mean density of BrdU + /Sox2 + cells among the total Sox2 + cells. Scale bars , 15 μm in C (applies to A – C ); 15 μm in D .

    Techniques Used: Immunostaining, Labeling, Mouse Assay

    Up-regulation of Sox2 expression in injured auditory nerve. A , B Real-time RT-PCR assays show fold changes for Sox2 and Hess5 mRNA expression in the auditory nerves taken from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears. Data are present as fold changes from control value ± standard error ( n = 3–5 per group). C Western blot assays with Sox2 antibody show a single 34-kDa band of endogenous Sox2 in normal, 3-, and 7-day post-treated ears. Expression levels of Sox2 increase after ouabain exposure, whereas the expression levels of NF 200 and β-tubulin decrease due to losses of SGNs in ouabain-treated ears. β-actin blotting was used to verify equal protein loading.
    Figure Legend Snippet: Up-regulation of Sox2 expression in injured auditory nerve. A , B Real-time RT-PCR assays show fold changes for Sox2 and Hess5 mRNA expression in the auditory nerves taken from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears. Data are present as fold changes from control value ± standard error ( n = 3–5 per group). C Western blot assays with Sox2 antibody show a single 34-kDa band of endogenous Sox2 in normal, 3-, and 7-day post-treated ears. Expression levels of Sox2 increase after ouabain exposure, whereas the expression levels of NF 200 and β-tubulin decrease due to losses of SGNs in ouabain-treated ears. β-actin blotting was used to verify equal protein loading.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    Sox2 + cells stain positively for neural glial marker in injured auditory nerve. A – C Dual immunostaining for S100 ( red ) and Sox2 ( green ) in the auditory nerve of a mouse 7 days after ouabain treatment. D – F Dual immunostaining for Sox10 ( red ) and Sox2 ( green ) in the mouse shown in A – C . Nuclei were counterstained with bis-benzimide ( blue ). Scale bar , 10 μm in F (applies to A – F ).
    Figure Legend Snippet: Sox2 + cells stain positively for neural glial marker in injured auditory nerve. A – C Dual immunostaining for S100 ( red ) and Sox2 ( green ) in the auditory nerve of a mouse 7 days after ouabain treatment. D – F Dual immunostaining for Sox10 ( red ) and Sox2 ( green ) in the mouse shown in A – C . Nuclei were counterstained with bis-benzimide ( blue ). Scale bar , 10 μm in F (applies to A – F ).

    Techniques Used: Staining, Marker, Immunostaining

    Increased Sox2 + cells within Rosenthal’s canal in injured auditory nerves. A – C Nuclear staining pattern of Sox2 antibody ( green ) in normal auditory nerve. PI nuclear counterstaining ( red ) reveals nuclear profiles of Sox2 + cells. The nuclei of SGNs are identified by their large spherical nuclei ( arrowheads ). Note that SGNs do not express or express a very low level of Sox2 and were not considered as Sox2 + cells in this study. D , E Dual immunostaining for Sox2 ( green ) and a neuronal marker, NF 200 ( NF , red ) reconfirms that high-intensity expression of Sox2 appears in the nuclei of Schwann- and or satellite-like cells ( arrows ). E Enlarged image of corresponding boxed area in D . F Sox2 + cells in the auditory nerve 7 days after ouabain treatment. An arrowhead indicates a surviving SGN process. G Mean density of Sox2 + cells within the Rosenthal’s canal in the apical, middle, and basal turns from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). Scale bars , 8 μm in C (applies to A – C ); 7 μm in D ; 7 μm in F (applies to E – F ).
    Figure Legend Snippet: Increased Sox2 + cells within Rosenthal’s canal in injured auditory nerves. A – C Nuclear staining pattern of Sox2 antibody ( green ) in normal auditory nerve. PI nuclear counterstaining ( red ) reveals nuclear profiles of Sox2 + cells. The nuclei of SGNs are identified by their large spherical nuclei ( arrowheads ). Note that SGNs do not express or express a very low level of Sox2 and were not considered as Sox2 + cells in this study. D , E Dual immunostaining for Sox2 ( green ) and a neuronal marker, NF 200 ( NF , red ) reconfirms that high-intensity expression of Sox2 appears in the nuclei of Schwann- and or satellite-like cells ( arrows ). E Enlarged image of corresponding boxed area in D . F Sox2 + cells in the auditory nerve 7 days after ouabain treatment. An arrowhead indicates a surviving SGN process. G Mean density of Sox2 + cells within the Rosenthal’s canal in the apical, middle, and basal turns from control, 1-, 3-, 7-, 14-, and 30-day post-treated ears ( n = 3–6 per group). Scale bars , 8 μm in C (applies to A – C ); 7 μm in D ; 7 μm in F (applies to E – F ).

    Techniques Used: Staining, Immunostaining, Marker, Expressing

    20) Product Images from "Adaptor Protein LNK Is a Negative Regulator of Brain Neural Stem Cell Proliferation after Stroke"

    Article Title: Adaptor Protein LNK Is a Negative Regulator of Brain Neural Stem Cell Proliferation after Stroke

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0474-12.2012

    LNK is expressed in SVZ NSPCs in vivo and in vitro . A , RT-PCR and Q-PCR analysis of Lnk expression in tissue and neurospheres derived from mouse SVZ, and in FAC-sorted neuroblasts (Dcx-GFP), NSPCs (Sox2-GFP), and microglia (Iba1-GFP) from SVZ and hippocampus (HPC) of reporter mice. B , Confocal photomicrographs showing LNK immunoreactivity in mouse SVZ neurospheres (NS) costained for SOX2. Double-positive cell depicted by arrowhead on low-magnification images is shown also at higher magnification. C , RT-PCR analysis of LNK expression in GE tissue and NS derived from human fetal GE (hGE). Photomicrographs showing LNK immunoreactivity in human NS (hNS) costained for SOX2. Arrowhead indicates example of double-labeled cell. D , RT-PCR analysis of LNK in three different specimens of adult human SVZ tissue. E , Overview of adult human SVZ stained for LNK (green) and GFAP (red). Boxed areas shown in higher magnification, top box ( F–I ) and lower box ( J–M ) depict LNK+ ( F , J ), GFAP+ ( G , K ), LNK+/GFAP+ ( H , I ), and Hoechst+ ( I , M ) cells. Arrowheads indicate double-labeled cells. Scale bars: B , 50 μm; (in C ) C–D , 20 μm; E , 15 μm, and (in M ) F–M , 5 μm.
    Figure Legend Snippet: LNK is expressed in SVZ NSPCs in vivo and in vitro . A , RT-PCR and Q-PCR analysis of Lnk expression in tissue and neurospheres derived from mouse SVZ, and in FAC-sorted neuroblasts (Dcx-GFP), NSPCs (Sox2-GFP), and microglia (Iba1-GFP) from SVZ and hippocampus (HPC) of reporter mice. B , Confocal photomicrographs showing LNK immunoreactivity in mouse SVZ neurospheres (NS) costained for SOX2. Double-positive cell depicted by arrowhead on low-magnification images is shown also at higher magnification. C , RT-PCR analysis of LNK expression in GE tissue and NS derived from human fetal GE (hGE). Photomicrographs showing LNK immunoreactivity in human NS (hNS) costained for SOX2. Arrowhead indicates example of double-labeled cell. D , RT-PCR analysis of LNK in three different specimens of adult human SVZ tissue. E , Overview of adult human SVZ stained for LNK (green) and GFAP (red). Boxed areas shown in higher magnification, top box ( F–I ) and lower box ( J–M ) depict LNK+ ( F , J ), GFAP+ ( G , K ), LNK+/GFAP+ ( H , I ), and Hoechst+ ( I , M ) cells. Arrowheads indicate double-labeled cells. Scale bars: B , 50 μm; (in C ) C–D , 20 μm; E , 15 μm, and (in M ) F–M , 5 μm.

    Techniques Used: In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Derivative Assay, Mouse Assay, Labeling, Staining

    Proliferation of NSPCs in SVZ is enhanced after stroke in Lnk −/− mice. Number and distribution of cells expressing p-H3 ( A , C ) and BrdU ( B , D ) in SVZ of intact WT and Lnk −/− mice ( A , B ) or 7 d after stroke ( C , D ). Representative confocal images of BrdU+/Dcx+ ( E ) and BrdU+/Sox2+ ( F ) cells in the SVZ after stroke. Number of proliferating Dcx+ and Sox2+ cells after stroke ( G ). Size and distribution of lesioned area after stroke ( H ). BrdU was injected 4 times 2 h apart in intact animals ( A , B ) or once daily following MCAO for 7 d ( C–F ). Ipsilateral denotes side of stroke. Means ± SEM, n = 4 and 5 for WT and Lnk −/− mice, respectively. * p
    Figure Legend Snippet: Proliferation of NSPCs in SVZ is enhanced after stroke in Lnk −/− mice. Number and distribution of cells expressing p-H3 ( A , C ) and BrdU ( B , D ) in SVZ of intact WT and Lnk −/− mice ( A , B ) or 7 d after stroke ( C , D ). Representative confocal images of BrdU+/Dcx+ ( E ) and BrdU+/Sox2+ ( F ) cells in the SVZ after stroke. Number of proliferating Dcx+ and Sox2+ cells after stroke ( G ). Size and distribution of lesioned area after stroke ( H ). BrdU was injected 4 times 2 h apart in intact animals ( A , B ) or once daily following MCAO for 7 d ( C–F ). Ipsilateral denotes side of stroke. Means ± SEM, n = 4 and 5 for WT and Lnk −/− mice, respectively. * p

    Techniques Used: Mouse Assay, Expressing, Injection

    21) Product Images from "Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity"

    Article Title: Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.09.019

    CD44+/CD24+ antigens identify TRTICs and GSCs. a. Flow cytometry plot showing the expression of CD24 and CD44 in OSU53GSC and PKAC2GSC cells after 10 Gy radiation treatment (n = 3). b. Phase contrast and GFP+/CD24+/CD44+ GFP cells showing sphere-forming ability at day1 to day15. Microscopic images of OSU53GSC and OSU68GSC CD44+/CD24+ and CD44-/CD24- TRTICs 10 days after sorting pure population (n = 3). c. Kaplan-Meier survival plots of NOD-SCID mice bearing tumors from CD24+/CD44+ and CD24-/CD44- populations of OSU53GSC and OSU68GSC cells (n = 3) (log-rank (Mantel-Cox) test). d. Immunohistochemistry of Sox2, Nestin, CD44, Hif-1α and VEGF in U87 TRTIC-derived tumors (n = 3). The Hematoxylin and Eosin stained tumor section demonstrates the adjacent normal tissue in the Sox2, Nestin and CD44 images. The Hif-1α and VEGF adjacent normal tissue is represented as individual boxes to the left of their respective tumor staining. e. Western blots showing the protein expression pattern of unsorted, CD44+/CD24+ and CD24-/CD44- OSU53GSC and OSU68GSC cells (n = 3). Scale bar = 50 μm.
    Figure Legend Snippet: CD44+/CD24+ antigens identify TRTICs and GSCs. a. Flow cytometry plot showing the expression of CD24 and CD44 in OSU53GSC and PKAC2GSC cells after 10 Gy radiation treatment (n = 3). b. Phase contrast and GFP+/CD24+/CD44+ GFP cells showing sphere-forming ability at day1 to day15. Microscopic images of OSU53GSC and OSU68GSC CD44+/CD24+ and CD44-/CD24- TRTICs 10 days after sorting pure population (n = 3). c. Kaplan-Meier survival plots of NOD-SCID mice bearing tumors from CD24+/CD44+ and CD24-/CD44- populations of OSU53GSC and OSU68GSC cells (n = 3) (log-rank (Mantel-Cox) test). d. Immunohistochemistry of Sox2, Nestin, CD44, Hif-1α and VEGF in U87 TRTIC-derived tumors (n = 3). The Hematoxylin and Eosin stained tumor section demonstrates the adjacent normal tissue in the Sox2, Nestin and CD44 images. The Hif-1α and VEGF adjacent normal tissue is represented as individual boxes to the left of their respective tumor staining. e. Western blots showing the protein expression pattern of unsorted, CD44+/CD24+ and CD24-/CD44- OSU53GSC and OSU68GSC cells (n = 3). Scale bar = 50 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Immunohistochemistry, Derivative Assay, Staining, Western Blot

    22) Product Images from "Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector"

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00415

    A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .
    Figure Legend Snippet: A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .

    Techniques Used: Injection, Expressing, Labeling, Marker

    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.
    Figure Legend Snippet: Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Techniques Used: Injection, Expressing, Labeling, Staining

    Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .
    Figure Legend Snippet: Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .

    Techniques Used: Labeling, In Vivo, In Vitro, Derivative Assay

    23) Product Images from "Lineage tracing of axial progenitors using Nkx1-2CreERT2 mice defines their trunk and tail contributions"

    Article Title: Lineage tracing of axial progenitors using Nkx1-2CreERT2 mice defines their trunk and tail contributions

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.164319

    A subset of Nkx1-2 -expressing cells and/or their progeny express SOX2 and T. (A) Transverse sections through the rostral node, NSB and CLE of an E8.5 Nkx1-2CreER T2 embryo that was exposed to tamoxifen at E7.5 and immunolabelled for SOX2, T and YFP ( n =7). (B) Transverse sections through the tail end of an E10.5 Nkx1-2CreER T2 embryo that was exposed to tamoxifen at E9.5 and immunolabelled for SOX2, T and YFP ( n =9). Abbreviations are as in Fig. 1 . nml, neuromesodermal lip; not*, notochord end. Scale bars: 100 µm.
    Figure Legend Snippet: A subset of Nkx1-2 -expressing cells and/or their progeny express SOX2 and T. (A) Transverse sections through the rostral node, NSB and CLE of an E8.5 Nkx1-2CreER T2 embryo that was exposed to tamoxifen at E7.5 and immunolabelled for SOX2, T and YFP ( n =7). (B) Transverse sections through the tail end of an E10.5 Nkx1-2CreER T2 embryo that was exposed to tamoxifen at E9.5 and immunolabelled for SOX2, T and YFP ( n =9). Abbreviations are as in Fig. 1 . nml, neuromesodermal lip; not*, notochord end. Scale bars: 100 µm.

    Techniques Used: Expressing

    SOX2 and T co-expression within Nkx1-2 regions. (A) Transverse sections across the rostral node, NSB and CLE of an E8.5 embryo immunolabelled for SOX2 and T ( n =4). (B) Transverse sections across the tail end of an E10.5 embryo immunolabelled for SOX2 and T ( n =7). The cartoons in (A) and (B) depict the expression pattern of Nkx1-2 (as shown in Fig. 1 ). The different levels of Nkx1-2 expression (based on in situ hybridisation signal) are represented by different grey intensities (light grey, low; dark grey, high). The dashed lines delineate regions not limited by basement membrane. Abbreviations are as in Fig. 1 . nml, neuromesodermal lip; not*, notochord end; som, somite. Scale bars: 50 µm.
    Figure Legend Snippet: SOX2 and T co-expression within Nkx1-2 regions. (A) Transverse sections across the rostral node, NSB and CLE of an E8.5 embryo immunolabelled for SOX2 and T ( n =4). (B) Transverse sections across the tail end of an E10.5 embryo immunolabelled for SOX2 and T ( n =7). The cartoons in (A) and (B) depict the expression pattern of Nkx1-2 (as shown in Fig. 1 ). The different levels of Nkx1-2 expression (based on in situ hybridisation signal) are represented by different grey intensities (light grey, low; dark grey, high). The dashed lines delineate regions not limited by basement membrane. Abbreviations are as in Fig. 1 . nml, neuromesodermal lip; not*, notochord end; som, somite. Scale bars: 50 µm.

    Techniques Used: Expressing, In Situ, Hybridization

    Lineage tracing of cells expressing Nkx1-2 at E10.5. (A,B) Timed-pregnant Nkx1-2CreER T2 mice received tamoxifen at E10.5 and the contribution of YFP + cells to developing embryos was assessed at E11.5 (A) and E12.5 (B). (A) Dorsal view (composite MIP) of the tail of an E11.5 embryo immunolabelled for YFP on whole-mount ( n =7). (Aa-Ad) Transverse sections representative of the levels indicated in A and immunolabelled for SOX2, T and YFP ( n =9). YFP + cells contributed to the secondary neural tube (Aa-Ad), presomitic mesoderm (Ab-Ad) and tail bud mesenchyme (Ad). (B) Side view (MIP) of the tail of an E12.5 embryo immunolabelled for YFP on whole-mount ( n =9). Abbreviations are as in Fig. 1 . vmes, ventral tail bud mesoderm. Scale bars: 100 µm (whole-mount embryos); 50 µm (transverse sections).
    Figure Legend Snippet: Lineage tracing of cells expressing Nkx1-2 at E10.5. (A,B) Timed-pregnant Nkx1-2CreER T2 mice received tamoxifen at E10.5 and the contribution of YFP + cells to developing embryos was assessed at E11.5 (A) and E12.5 (B). (A) Dorsal view (composite MIP) of the tail of an E11.5 embryo immunolabelled for YFP on whole-mount ( n =7). (Aa-Ad) Transverse sections representative of the levels indicated in A and immunolabelled for SOX2, T and YFP ( n =9). YFP + cells contributed to the secondary neural tube (Aa-Ad), presomitic mesoderm (Ab-Ad) and tail bud mesenchyme (Ad). (B) Side view (MIP) of the tail of an E12.5 embryo immunolabelled for YFP on whole-mount ( n =9). Abbreviations are as in Fig. 1 . vmes, ventral tail bud mesoderm. Scale bars: 100 µm (whole-mount embryos); 50 µm (transverse sections).

    Techniques Used: Expressing, Mouse Assay

    24) Product Images from "Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells"

    Article Title: Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2011.054

    Differentiation protocol implemented for the generation of DA neurons and validation of specific expression of LMX1A in neural precursor cells. (a) Schematic representation of the different stages for the in vitro differentiation of pluripotent stem cells toward DA neurons. At Stage 1, EBs are formed by aggregation. At Stage 2, NPCs are obtained culturing the EBs in suspension with N2B27 medium supplemented with FGF2, FGF8, and SHH. At Stage 3, NPCs are cocultured with PA6 for 3 weeks and DA neurons are generated. (b) NPCs formed at Stage 2 express the neural precursor markers NESTIN (c) and SOX2 (d) . (e) At Stage 3, some of the TUJ1-positive cells generated coexpress TH. (f) Omitting the EB generation step or the coculture with PA6 cells results in
    Figure Legend Snippet: Differentiation protocol implemented for the generation of DA neurons and validation of specific expression of LMX1A in neural precursor cells. (a) Schematic representation of the different stages for the in vitro differentiation of pluripotent stem cells toward DA neurons. At Stage 1, EBs are formed by aggregation. At Stage 2, NPCs are obtained culturing the EBs in suspension with N2B27 medium supplemented with FGF2, FGF8, and SHH. At Stage 3, NPCs are cocultured with PA6 for 3 weeks and DA neurons are generated. (b) NPCs formed at Stage 2 express the neural precursor markers NESTIN (c) and SOX2 (d) . (e) At Stage 3, some of the TUJ1-positive cells generated coexpress TH. (f) Omitting the EB generation step or the coculture with PA6 cells results in

    Techniques Used: Expressing, In Vitro, Generated

    Generation of hESC clones overexpressing LMX1A . (a) Southern blot analysis of clones D, G, and L using a probe against LMX1A informing about the lentiviral integration copy number. Integration results using qPCR confirm the results obtained in the Southern blot. (b) The transduced clone D showed normal karyotype. (c–e) After the infection, clone D retains the pluripotency capacity as judged by the expression of pluripotency markers NANOG, TRA-1-81, OCT-4, SSEA-3, SOX2, and SSEA-4. The ability to give rise to the three germ layers in vitro (f–h) and in vivo (i–k)
    Figure Legend Snippet: Generation of hESC clones overexpressing LMX1A . (a) Southern blot analysis of clones D, G, and L using a probe against LMX1A informing about the lentiviral integration copy number. Integration results using qPCR confirm the results obtained in the Southern blot. (b) The transduced clone D showed normal karyotype. (c–e) After the infection, clone D retains the pluripotency capacity as judged by the expression of pluripotency markers NANOG, TRA-1-81, OCT-4, SSEA-3, SOX2, and SSEA-4. The ability to give rise to the three germ layers in vitro (f–h) and in vivo (i–k)

    Techniques Used: Clone Assay, Southern Blot, Real-time Polymerase Chain Reaction, Infection, Expressing, In Vitro, In Vivo

    25) Product Images from "Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation"

    Article Title: Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2013.00166

    Shh controls the pool of RG cells (GFAP+, Sox2+, GLAST+) that express EGFR. (A) Western blot and densitometry analysis of EGFR show higher levels of EGFR in Shh-treated explants in comparison to samples treated with bFGF and positive control EGF. (B) Flow cytometry of cells harvested from E18.5 cortical explants. Treatment for 48 h with Cyc (10 μM) provokes similar decreases in the pool of EGFR- and GFAP-expressing cells. Histograms are representative of three independent experiments expressed as the percentage of the mean ± and SEM ( ** p
    Figure Legend Snippet: Shh controls the pool of RG cells (GFAP+, Sox2+, GLAST+) that express EGFR. (A) Western blot and densitometry analysis of EGFR show higher levels of EGFR in Shh-treated explants in comparison to samples treated with bFGF and positive control EGF. (B) Flow cytometry of cells harvested from E18.5 cortical explants. Treatment for 48 h with Cyc (10 μM) provokes similar decreases in the pool of EGFR- and GFAP-expressing cells. Histograms are representative of three independent experiments expressed as the percentage of the mean ± and SEM ( ** p

    Techniques Used: Western Blot, Positive Control, Flow Cytometry, Cytometry, Expressing

    26) Product Images from "Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro"

    Article Title: Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.166215

    RT-qPCR for selected genes during dSMADi-RA differentiation and generation of a GFP-Nkx1.2 reporter line. (A-E) RT-qPCR assessing relative expression of key marker genes in H9 cells exposed to the dSMADi-RA protocol ( Fig. 1 F). (A) Declining expression of the pluripotency genes OCT4 and NANOG . (B) SOX2 , BRA ( T ) and CDX2 expression dynamics. (C) HOXB4 and HOXC6 during differentiation. (D) Expression of the neural progenitor marker PAX6 . (E) WNT8A / C and NKX1 . 2 , which are characteristic of preneural progenitors and NMPs. **** P
    Figure Legend Snippet: RT-qPCR for selected genes during dSMADi-RA differentiation and generation of a GFP-Nkx1.2 reporter line. (A-E) RT-qPCR assessing relative expression of key marker genes in H9 cells exposed to the dSMADi-RA protocol ( Fig. 1 F). (A) Declining expression of the pluripotency genes OCT4 and NANOG . (B) SOX2 , BRA ( T ) and CDX2 expression dynamics. (C) HOXB4 and HOXC6 during differentiation. (D) Expression of the neural progenitor marker PAX6 . (E) WNT8A / C and NKX1 . 2 , which are characteristic of preneural progenitors and NMPs. **** P

    Techniques Used: Quantitative RT-PCR, Expressing, Marker

    Protocol for neural differentiation of human NMP-like cells. (A) Schematic of mouse E8.5 caudal embryo. Selected progenitor cell marker genes and signalling pathways operating during posterior neural differentiation. (B,B′) Schematic of the developed differentiation protocol, including a dual-SMAD inhibition step (dSMADi-RA) (B), and immunocytochemistry for Bra (T) and Sox2 in day 3 NMPs (three independent experiments) (B′). (C) RT-qPCR showing PAX6 in the H9 cell line differentiated as in B, with or without 100 nM RA from day 3. (D) RT-qPCR for PAX6 in cells differentiated as in B, with varying SMAD inhibitor inclusion day 2-4. RT-qPCR graphs represent expression normalized to GAPDH and relative to hESC levels (three independent experiments, error bars indicate the s.e.m.; **** P
    Figure Legend Snippet: Protocol for neural differentiation of human NMP-like cells. (A) Schematic of mouse E8.5 caudal embryo. Selected progenitor cell marker genes and signalling pathways operating during posterior neural differentiation. (B,B′) Schematic of the developed differentiation protocol, including a dual-SMAD inhibition step (dSMADi-RA) (B), and immunocytochemistry for Bra (T) and Sox2 in day 3 NMPs (three independent experiments) (B′). (C) RT-qPCR showing PAX6 in the H9 cell line differentiated as in B, with or without 100 nM RA from day 3. (D) RT-qPCR for PAX6 in cells differentiated as in B, with varying SMAD inhibitor inclusion day 2-4. RT-qPCR graphs represent expression normalized to GAPDH and relative to hESC levels (three independent experiments, error bars indicate the s.e.m.; **** P

    Techniques Used: Marker, Inhibition, Immunocytochemistry, Quantitative RT-PCR, Expressing

    27) Product Images from "Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro"

    Article Title: Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.166215

    Protocol for neural differentiation of human NMP-like cells. (A) Schematic of mouse E8.5 caudal embryo. Selected progenitor cell marker genes and signalling pathways operating during posterior neural differentiation. (B,B′) Schematic of the developed differentiation protocol, including a dual-SMAD inhibition step (dSMADi-RA) (B), and immunocytochemistry for Bra (T) and Sox2 in day 3 NMPs (three independent experiments) (B′). (C) RT-qPCR showing PAX6 in the H9 cell line differentiated as in B, with or without 100 nM RA from day 3. (D) RT-qPCR for PAX6 in cells differentiated as in B, with varying SMAD inhibitor inclusion day 2-4. RT-qPCR graphs represent expression normalized to GAPDH and relative to hESC levels (three independent experiments, error bars indicate the s.e.m.; **** P
    Figure Legend Snippet: Protocol for neural differentiation of human NMP-like cells. (A) Schematic of mouse E8.5 caudal embryo. Selected progenitor cell marker genes and signalling pathways operating during posterior neural differentiation. (B,B′) Schematic of the developed differentiation protocol, including a dual-SMAD inhibition step (dSMADi-RA) (B), and immunocytochemistry for Bra (T) and Sox2 in day 3 NMPs (three independent experiments) (B′). (C) RT-qPCR showing PAX6 in the H9 cell line differentiated as in B, with or without 100 nM RA from day 3. (D) RT-qPCR for PAX6 in cells differentiated as in B, with varying SMAD inhibitor inclusion day 2-4. RT-qPCR graphs represent expression normalized to GAPDH and relative to hESC levels (three independent experiments, error bars indicate the s.e.m.; **** P

    Techniques Used: Marker, Inhibition, Immunocytochemistry, Quantitative RT-PCR, Expressing

    28) Product Images from "The nuclear periphery of embryonic stem cells is a transcriptionally permissive and repressive compartment"

    Article Title: The nuclear periphery of embryonic stem cells is a transcriptionally permissive and repressive compartment

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.052555

    Transcribing MMU14 loci associate closely with the nuclear periphery. (A) Map of genes (black bars, left) and gene `deserts' in the MMU14 region under study. Sce1, Slain1, Rbm26 and Ndfip2 ). BACs used for FISH probes are indicated on the right. (B,C) Two-color FISH detects Sce1 and Slain1 (green) and Rbm26 and Ndfip2 (red) near the lamina (blue) of ESCs. The cell in B shows three of these four loci (arrows) contacting the lamina (inset, white). Loci from the same chromosome are separated by approximately 1 > m. An X - Y optical section and sections from X - Z projections are shown. For the cell in C, a maximum X - Z projection and single X - Z and Y - Z sections are shown. (D) 3D distributions of Sce1 and Slain1 (green), Rbm26 and Ndfip2 (red), and LMNB1 (black) were measured by Erosion for 97 ESCs. Both active loci are most often 200-400 nm from the peak lamin signal. (E) MMU14 gene positions (green) contrast with that of active Sox2 (red) on MMU3 ( n =93). (F,G) MMU14 gene positions (green) are similar to those of two MMU14 gene deserts (red; n =97 and 99, respectively). In D-G, mean shell volumes relative to the total nuclear volume are shown (gray, arbitrary units). (H,I) RNA-FISH detects Rbm26 and Ndfip2 transcripts (red) adjacent to the nuclear lamina (anti-LMNB1, green) in two ESC nuclei. Single X - Y, X - Z and Y - Z sections of both FISH signals in each cell are shown as indicated. Scale bars: 1 > m. (J) Erosion analysis of 93 ESCs shows a peak of Rbm26 and Ndfip2 transcript signal 400 nm from the lamina, similar to the locus probed by DNA-FISH (D). Error bars, s.e.m.
    Figure Legend Snippet: Transcribing MMU14 loci associate closely with the nuclear periphery. (A) Map of genes (black bars, left) and gene `deserts' in the MMU14 region under study. Sce1, Slain1, Rbm26 and Ndfip2 ). BACs used for FISH probes are indicated on the right. (B,C) Two-color FISH detects Sce1 and Slain1 (green) and Rbm26 and Ndfip2 (red) near the lamina (blue) of ESCs. The cell in B shows three of these four loci (arrows) contacting the lamina (inset, white). Loci from the same chromosome are separated by approximately 1 > m. An X - Y optical section and sections from X - Z projections are shown. For the cell in C, a maximum X - Z projection and single X - Z and Y - Z sections are shown. (D) 3D distributions of Sce1 and Slain1 (green), Rbm26 and Ndfip2 (red), and LMNB1 (black) were measured by Erosion for 97 ESCs. Both active loci are most often 200-400 nm from the peak lamin signal. (E) MMU14 gene positions (green) contrast with that of active Sox2 (red) on MMU3 ( n =93). (F,G) MMU14 gene positions (green) are similar to those of two MMU14 gene deserts (red; n =97 and 99, respectively). In D-G, mean shell volumes relative to the total nuclear volume are shown (gray, arbitrary units). (H,I) RNA-FISH detects Rbm26 and Ndfip2 transcripts (red) adjacent to the nuclear lamina (anti-LMNB1, green) in two ESC nuclei. Single X - Y, X - Z and Y - Z sections of both FISH signals in each cell are shown as indicated. Scale bars: 1 > m. (J) Erosion analysis of 93 ESCs shows a peak of Rbm26 and Ndfip2 transcript signal 400 nm from the lamina, similar to the locus probed by DNA-FISH (D). Error bars, s.e.m.

    Techniques Used: Fluorescence In Situ Hybridization

    29) Product Images from "Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis"

    Article Title: Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.123141

    Ectopic K8+ cells express Atoh1, Sox2 and Isl1, some express caspase-3, but none is mitotically active. Experimental induction paradigm is shown at the top of the figure. (A-H‴) Sectioned back skin of adolescent K14 Cre ; ROSA rtTA ; Tet Atoh1 mice showing ectopic K8+ cells in hair follicles four days (A-C‴,E-E‴,G-G‴) and 2 weeks (D-D‴,F-F‴,H-H‴) post-induction. Sections were co-immunostained for cleaved caspase-3 (A′), Ki67 (B′) Atoh1, (C′,D′), Sox2 (E′,F′), Isl1 (G′,H′) and K8 (A″,B″,C″,D″,E″,F″,G″,H″). Yellow arrows indicate double-positive cells. Red arrows indicate K8+ cells not expressing the marker of interest. All images were taken using the same exposure settings, demonstrating greater fluorescence intensity of each transcription factor at 4 days versus 2 weeks post-doxycycline. The average percentage of ectopic K8+ cells (±s.e.m.) co-expressing each marker is shown ( n =100-200 K8+ cells from each of three mice). Scale bars: 10 μm.
    Figure Legend Snippet: Ectopic K8+ cells express Atoh1, Sox2 and Isl1, some express caspase-3, but none is mitotically active. Experimental induction paradigm is shown at the top of the figure. (A-H‴) Sectioned back skin of adolescent K14 Cre ; ROSA rtTA ; Tet Atoh1 mice showing ectopic K8+ cells in hair follicles four days (A-C‴,E-E‴,G-G‴) and 2 weeks (D-D‴,F-F‴,H-H‴) post-induction. Sections were co-immunostained for cleaved caspase-3 (A′), Ki67 (B′) Atoh1, (C′,D′), Sox2 (E′,F′), Isl1 (G′,H′) and K8 (A″,B″,C″,D″,E″,F″,G″,H″). Yellow arrows indicate double-positive cells. Red arrows indicate K8+ cells not expressing the marker of interest. All images were taken using the same exposure settings, demonstrating greater fluorescence intensity of each transcription factor at 4 days versus 2 weeks post-doxycycline. The average percentage of ectopic K8+ cells (±s.e.m.) co-expressing each marker is shown ( n =100-200 K8+ cells from each of three mice). Scale bars: 10 μm.

    Techniques Used: Mouse Assay, Expressing, Marker, Fluorescence

    30) Product Images from "A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells"

    Article Title: A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2009.03.026

    ES cell-derived endoderm acquire foregut cell fates ( A–J’ and P–Z’ ) OS25 (Sox2 β geo /+ ) or ( K–O ) Pdx1 LacZ /+ cells were cultured for 5 days in 100 ng/ml activin and subsequently treated with 5 ng/ml Wnt3a, 10 ng/ml FGF4 and/or 0,1 µM RA for 3 days as indicated. The co-expression of Foxa2 and Sox2 (foregut), Pdx1 (midgut) or Cdx2 (hindgut) was analysed by immunofluorescence. The expression of Sox2 ( A–E ) and Pdx1 ( K–O ) was confirmed by analysing β-galactosidase activity using X-gal staining.
    Figure Legend Snippet: ES cell-derived endoderm acquire foregut cell fates ( A–J’ and P–Z’ ) OS25 (Sox2 β geo /+ ) or ( K–O ) Pdx1 LacZ /+ cells were cultured for 5 days in 100 ng/ml activin and subsequently treated with 5 ng/ml Wnt3a, 10 ng/ml FGF4 and/or 0,1 µM RA for 3 days as indicated. The co-expression of Foxa2 and Sox2 (foregut), Pdx1 (midgut) or Cdx2 (hindgut) was analysed by immunofluorescence. The expression of Sox2 ( A–E ) and Pdx1 ( K–O ) was confirmed by analysing β-galactosidase activity using X-gal staining.

    Techniques Used: Derivative Assay, Cell Culture, Expressing, Immunofluorescence, Activity Assay, Staining

    31) Product Images from "In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm IdentityDevelopment of Spinal Cord Neurons in Delicate Balance"

    Article Title: In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm IdentityDevelopment of Spinal Cord Neurons in Delicate Balance

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001937

    Generation of NMPs from EpiSCs. (A) Brachyury/Sox2 immunocytochemistry in EpiSC cultures treated with FGF/CHIR for 72 h. (B) qPCR analysis for indicated markers in mouse EpiSCs treated with FGF/CHIR. Error bars = s.d. (n = 3). n/d, not determined. Results are represented as log 10 ratio of expression versus untreated EpiSCs. The data used to generate the plot can be found in Data S4 . (C) Combined fluorescence/brightfield microscopy showing donor cell incorporation of grafted GFP + EpiSC differentiated for 48 h in FGF/CHIR after 48 h embryo culture. (D) Table summarizing the incorporation of grafted GFP + EpiSC differentiated for 24 h or 48 h in Fgf/Wnt within host embryos. NT, neural tube; Som, somite; PSM, presomitic mesoderm; n/a, not applicable. (E) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Cell nuclei were stained with DAPI (blue). White boxes indicate the position of magnified images of GFP + cells.
    Figure Legend Snippet: Generation of NMPs from EpiSCs. (A) Brachyury/Sox2 immunocytochemistry in EpiSC cultures treated with FGF/CHIR for 72 h. (B) qPCR analysis for indicated markers in mouse EpiSCs treated with FGF/CHIR. Error bars = s.d. (n = 3). n/d, not determined. Results are represented as log 10 ratio of expression versus untreated EpiSCs. The data used to generate the plot can be found in Data S4 . (C) Combined fluorescence/brightfield microscopy showing donor cell incorporation of grafted GFP + EpiSC differentiated for 48 h in FGF/CHIR after 48 h embryo culture. (D) Table summarizing the incorporation of grafted GFP + EpiSC differentiated for 24 h or 48 h in Fgf/Wnt within host embryos. NT, neural tube; Som, somite; PSM, presomitic mesoderm; n/a, not applicable. (E) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Cell nuclei were stained with DAPI (blue). White boxes indicate the position of magnified images of GFP + cells.

    Techniques Used: Immunocytochemistry, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Embryo Culture, Immunofluorescence, Staining

    Transient Wnt and FGF signalling induce dual fated neuromesodermal progenitors. (A) Schematic of differentiation protocols used to generate mesoderm and neural cells from a common NM progenitor population. (B) mRNA-seq expression values of Sox2 , Brachyury , Tbx6 and Cdx2 following exposure to bFGF alone or bFGF/CHIR for 12 h (D2.5) and 24 h (D3). Activation of Wnt signalling with CHIR upregulated Brachyury within 12 h. Expression of Tbx6 and Cdx2 was also upregulated in NMPs by D3, whereas Sox2 transcript levels were decreased. (C) Immunostaining of cells treated with FGF/Wnt revealed the coexpression of Brachyury with Sox2 (NMPs). In the absence of Wnt, NPCs express Sox2 but the expression of Brachyury is only evident in a very small proportion of cells. (D) mRNA expression values of neural ( Sox1 , Sox2 , Sox3 ) and mesodermal progenitors markers ( Tbx6 , Bra , Msgn1 ) in posterior neural (N P ) and mesodermal cells (Meso) at D5 show the generation of distinct populations depending on treatment after D3. Removal of Wnt at D3 results in the generation of neural cells expressing Sox1–3 whereas continued Wnt exposure induces expression of Tbx6, Brachyury and Msgn1, characteristic of paraxial mesodermal. (E) Immunostaining indicates that continued Wnt exposure generates paraxial mesodermal progenitors that express Tbx6 at D5 and Desmin and MyoD at D8. (F) Sketch of a chick embryo (HH8–9) showing the injection site (IS) of NMP or N A cells. (G) NMP cells were labelled with DiI and transplanted in the CLE region. After 24 h the cells had incorporated into both the neural tube and somites. Whole-mount and transverse sections of HH17 chick embryos show the incorporation (asterisks) in the neural tube (H) and somites (I). (J) Table summarizing the number of chick embryos that were injected at stage HH8–9 and had engrafted cells in the neural tube, the somites or both 24 h later. Injection of N A cells resulted in incorporation only in the neural tube (K, L). All data used to generate the plots of Figure 2 can be found in Data S2 .
    Figure Legend Snippet: Transient Wnt and FGF signalling induce dual fated neuromesodermal progenitors. (A) Schematic of differentiation protocols used to generate mesoderm and neural cells from a common NM progenitor population. (B) mRNA-seq expression values of Sox2 , Brachyury , Tbx6 and Cdx2 following exposure to bFGF alone or bFGF/CHIR for 12 h (D2.5) and 24 h (D3). Activation of Wnt signalling with CHIR upregulated Brachyury within 12 h. Expression of Tbx6 and Cdx2 was also upregulated in NMPs by D3, whereas Sox2 transcript levels were decreased. (C) Immunostaining of cells treated with FGF/Wnt revealed the coexpression of Brachyury with Sox2 (NMPs). In the absence of Wnt, NPCs express Sox2 but the expression of Brachyury is only evident in a very small proportion of cells. (D) mRNA expression values of neural ( Sox1 , Sox2 , Sox3 ) and mesodermal progenitors markers ( Tbx6 , Bra , Msgn1 ) in posterior neural (N P ) and mesodermal cells (Meso) at D5 show the generation of distinct populations depending on treatment after D3. Removal of Wnt at D3 results in the generation of neural cells expressing Sox1–3 whereas continued Wnt exposure induces expression of Tbx6, Brachyury and Msgn1, characteristic of paraxial mesodermal. (E) Immunostaining indicates that continued Wnt exposure generates paraxial mesodermal progenitors that express Tbx6 at D5 and Desmin and MyoD at D8. (F) Sketch of a chick embryo (HH8–9) showing the injection site (IS) of NMP or N A cells. (G) NMP cells were labelled with DiI and transplanted in the CLE region. After 24 h the cells had incorporated into both the neural tube and somites. Whole-mount and transverse sections of HH17 chick embryos show the incorporation (asterisks) in the neural tube (H) and somites (I). (J) Table summarizing the number of chick embryos that were injected at stage HH8–9 and had engrafted cells in the neural tube, the somites or both 24 h later. Injection of N A cells resulted in incorporation only in the neural tube (K, L). All data used to generate the plots of Figure 2 can be found in Data S2 .

    Techniques Used: Expressing, Activation Assay, Immunostaining, Injection

    Generation and characterisation of hNMPs. (A) Scheme describing the culture conditions employed for neural differentiation of hES cells treated for 72 h either with FGF/CHIR or subjected to dual SMAD inhibition (LDN, LDN193189; SB43, SB431542). (B) BRACHYURY/SOX2 immunocytochemistry in undifferentiated and FGF/CHIR-treated (48 h) hES cells. Corresponding graphs depict image analysis of BRACHYURY and SOX2 expression in the indicated culture conditions. Numbers: percentages of cells in each quadrant. (C) qPCR analysis for indicated markers in hES cells treated with FGF/CHIR for 72 h (D3) or 96 h (D4). Error bars = s.d. (n = 2). Results are represented as log 10 ratio of expression versus untreated hES cells. (D) qPCR analysis for indicated differentiation markers in hES cells differentiated in N2B27 following either an NM progenitor induction- (N P ) or a dual SMAD inhibition-intermediate step (N A ). Error bars = s.d. (n = 2). Anterior, anterior neural markers; PXM, paraxial mesoderm; n/d, not determined. (E) Immunocytochemistry for SOX2/HOXC8 in N A and N P culture conditions indicated in the scheme (A). (F) Quantitation of the coexpression of Hoxc8 with Sox2 in N A and N P conditions. All data used to generate the plots can be found in Data S5 .
    Figure Legend Snippet: Generation and characterisation of hNMPs. (A) Scheme describing the culture conditions employed for neural differentiation of hES cells treated for 72 h either with FGF/CHIR or subjected to dual SMAD inhibition (LDN, LDN193189; SB43, SB431542). (B) BRACHYURY/SOX2 immunocytochemistry in undifferentiated and FGF/CHIR-treated (48 h) hES cells. Corresponding graphs depict image analysis of BRACHYURY and SOX2 expression in the indicated culture conditions. Numbers: percentages of cells in each quadrant. (C) qPCR analysis for indicated markers in hES cells treated with FGF/CHIR for 72 h (D3) or 96 h (D4). Error bars = s.d. (n = 2). Results are represented as log 10 ratio of expression versus untreated hES cells. (D) qPCR analysis for indicated differentiation markers in hES cells differentiated in N2B27 following either an NM progenitor induction- (N P ) or a dual SMAD inhibition-intermediate step (N A ). Error bars = s.d. (n = 2). Anterior, anterior neural markers; PXM, paraxial mesoderm; n/d, not determined. (E) Immunocytochemistry for SOX2/HOXC8 in N A and N P culture conditions indicated in the scheme (A). (F) Quantitation of the coexpression of Hoxc8 with Sox2 in N A and N P conditions. All data used to generate the plots can be found in Data S5 .

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

    Brachyury is necessary for mesoderm formation but not posterior neural identity. (A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of Tbx6 , Cdx2 and Hoxb1 relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra −/− ) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while Cdx2 and Hoxb1 continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra −/− ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes Hoxc8 and Hoxc9 are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers Sox1 and Sox2 are expressed only in Bra −/− cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra −/− and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra −/− cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra −/− cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N P ) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log 2+ scale). All data used for the plots can be found in Data S6 . (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.
    Figure Legend Snippet: Brachyury is necessary for mesoderm formation but not posterior neural identity. (A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of Tbx6 , Cdx2 and Hoxb1 relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra −/− ) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while Cdx2 and Hoxb1 continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra −/− ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes Hoxc8 and Hoxc9 are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers Sox1 and Sox2 are expressed only in Bra −/− cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra −/− and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra −/− cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra −/− cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N P ) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log 2+ scale). All data used for the plots can be found in Data S6 . (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.

    Techniques Used: Quantitative RT-PCR, Expressing, Activation Assay, Marker, Immunostaining, Cell Culture

    32) Product Images from "Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease"

    Article Title: Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201200215

    Generation and characterization of PD patient-specific iPSC lines A-C. Representative colonies of passage-20 LRRK2-PD-iPSC (cell line SP13.4) stained positive for the pluripotency-associated markers NANOG, OCT4 and SOX2 (green), TRA-1-81, SSEA3 and SSEA4 (red). D-F. Immunofluorescence analyses of LRRK2-PD-iPSC (cell line SP13.4) differentiated in vitro show the potential to generate cell derivatives of all three primary germ cell layers including ectoderm (D, stained for TUJ1, green), endoderm (E, stained for α-fetoprotein, green, and FOXA2, red) and mesoderm (F, stained for smooth muscle actin, SMA, red). G-I. Immunofluorescence analyses of sections from a teratoma induced by injecting LRRK2-PD-iPSC (cell line SP13.4), showing derivatives of the three main embryo germ layers: ectoderm (G, stained for TUJ1, green, and GFAP, red), endoderm (H, stained for α-fetoprotein, green, and FOXA2, red) and mesoderm (I, stained for SOX9, green, and chondroitin sulphate, CS, red). In ( A–I ) nuclei are counterstained with DAPI, shown in blue. Scale bars, 50 µm. J. LRRK2-PD-iPSC (cell line SP13.4) stained for alkaline phosphatase (AP) activity. K. Normal karyotype of LRRK2-PD-iPSC (cell line SP13.4) at passage 20. L. Bisulphite genomic sequencing of the OCT4 and NANOG promoters showing demethylation in LRRK2-PD-iPSC (cell line SP13.4). M. Southern blot analysis of LRRK2-PD-iPSC (cell line SP13.4) showing genomic integrations (asterisk) of the indicated retroviruses. N. RT-qPCR analyses of the expression levels of retroviral-derived reprogramming factors (transgenic) and endogenous expression levels (endogenous) of the indicated genes in LRRK2-PD-iPSC (cell line SP13.4). O. Direct sequence of genomic DNA from LRRK2-PD-iPSC (cell line SP13.4) identifying the LRRK2 G2019S mutation.
    Figure Legend Snippet: Generation and characterization of PD patient-specific iPSC lines A-C. Representative colonies of passage-20 LRRK2-PD-iPSC (cell line SP13.4) stained positive for the pluripotency-associated markers NANOG, OCT4 and SOX2 (green), TRA-1-81, SSEA3 and SSEA4 (red). D-F. Immunofluorescence analyses of LRRK2-PD-iPSC (cell line SP13.4) differentiated in vitro show the potential to generate cell derivatives of all three primary germ cell layers including ectoderm (D, stained for TUJ1, green), endoderm (E, stained for α-fetoprotein, green, and FOXA2, red) and mesoderm (F, stained for smooth muscle actin, SMA, red). G-I. Immunofluorescence analyses of sections from a teratoma induced by injecting LRRK2-PD-iPSC (cell line SP13.4), showing derivatives of the three main embryo germ layers: ectoderm (G, stained for TUJ1, green, and GFAP, red), endoderm (H, stained for α-fetoprotein, green, and FOXA2, red) and mesoderm (I, stained for SOX9, green, and chondroitin sulphate, CS, red). In ( A–I ) nuclei are counterstained with DAPI, shown in blue. Scale bars, 50 µm. J. LRRK2-PD-iPSC (cell line SP13.4) stained for alkaline phosphatase (AP) activity. K. Normal karyotype of LRRK2-PD-iPSC (cell line SP13.4) at passage 20. L. Bisulphite genomic sequencing of the OCT4 and NANOG promoters showing demethylation in LRRK2-PD-iPSC (cell line SP13.4). M. Southern blot analysis of LRRK2-PD-iPSC (cell line SP13.4) showing genomic integrations (asterisk) of the indicated retroviruses. N. RT-qPCR analyses of the expression levels of retroviral-derived reprogramming factors (transgenic) and endogenous expression levels (endogenous) of the indicated genes in LRRK2-PD-iPSC (cell line SP13.4). O. Direct sequence of genomic DNA from LRRK2-PD-iPSC (cell line SP13.4) identifying the LRRK2 G2019S mutation.

    Techniques Used: Staining, Immunofluorescence, In Vitro, Activity Assay, Genomic Sequencing, Southern Blot, Quantitative RT-PCR, Expressing, Derivative Assay, Transgenic Assay, Sequencing, Mutagenesis

    33) Product Images from "Double minute amplification of mutant PDGF receptor α in a mouse glioma model"

    Article Title: Double minute amplification of mutant PDGF receptor α in a mouse glioma model

    Journal: Scientific Reports

    doi: 10.1038/srep08468

    PDGFRα driven brain tumors display features of high grade glioma. (a–g) Histopathological analysis of tumor areas by H E staining shows a high concentration of mitotic figures (a, arrows), high cellularity and nuclear atypia (b), perineuronal satellitosis (c; N, neuronal nuclei), perivascular growth (d), intrafascicular growth (e), subarachnoid spreading (f), and areas of incipient necrosis (g; arrows point to pyknotic nuclei). (h–k) IF labeling of brain tumor sections for cell type specific markers. Nuclei labeled with DAPI are shown in blue. Tumor cells with high PDGFRα expression were highly proliferative, as seen by proliferation marker Ki67 (h), and express the OPC cell lineage markers Olig2, Sox2, Sox10, and Ng2, as well as the neural stem cell marker Nestin (i–k). Tumor cells were negative for immunosignal of astroglial marker GFAP, mature oligodendrocyte marker APC-CC1, and neuronal marker NeuN (l–n). Scale bars: 10 μm (a–g), 20 μm (h–n).
    Figure Legend Snippet: PDGFRα driven brain tumors display features of high grade glioma. (a–g) Histopathological analysis of tumor areas by H E staining shows a high concentration of mitotic figures (a, arrows), high cellularity and nuclear atypia (b), perineuronal satellitosis (c; N, neuronal nuclei), perivascular growth (d), intrafascicular growth (e), subarachnoid spreading (f), and areas of incipient necrosis (g; arrows point to pyknotic nuclei). (h–k) IF labeling of brain tumor sections for cell type specific markers. Nuclei labeled with DAPI are shown in blue. Tumor cells with high PDGFRα expression were highly proliferative, as seen by proliferation marker Ki67 (h), and express the OPC cell lineage markers Olig2, Sox2, Sox10, and Ng2, as well as the neural stem cell marker Nestin (i–k). Tumor cells were negative for immunosignal of astroglial marker GFAP, mature oligodendrocyte marker APC-CC1, and neuronal marker NeuN (l–n). Scale bars: 10 μm (a–g), 20 μm (h–n).

    Techniques Used: Staining, Concentration Assay, Labeling, Expressing, Marker

    34) Product Images from "mTOR regulates brain morphogenesis by mediating GSK3 signaling"

    Article Title: mTOR regulates brain morphogenesis by mediating GSK3 signaling

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.108282

    Expression and activity of mTOR-signaling components in the developing brain. (A) Top: mTOR signaling components were broadly expressed in the developing brain at E13.5. Bottom: phosphorylated forms of mTOR components were concentrated near the ventricular surface (arrows). Scale bar: 60 μm. (B) mTOR signaling was activated in Sox2-positive neural progenitors at the ventricular surface of E13.5 brain, as indicated by phospho-S6K expression. Scale bar: 20 μm. (C) Higher magnification images of cells immunostained with antibodies to mTOR, p-4EBP1 and p-S6. Dashed and dotted lines indicate a single cell and a nucleus, respectively.
    Figure Legend Snippet: Expression and activity of mTOR-signaling components in the developing brain. (A) Top: mTOR signaling components were broadly expressed in the developing brain at E13.5. Bottom: phosphorylated forms of mTOR components were concentrated near the ventricular surface (arrows). Scale bar: 60 μm. (B) mTOR signaling was activated in Sox2-positive neural progenitors at the ventricular surface of E13.5 brain, as indicated by phospho-S6K expression. Scale bar: 20 μm. (C) Higher magnification images of cells immunostained with antibodies to mTOR, p-4EBP1 and p-S6. Dashed and dotted lines indicate a single cell and a nucleus, respectively.

    Techniques Used: Expressing, Activity Assay

    35) Product Images from "Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background"

    Article Title: Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.057000

    Early neoplasias express high levels of ECAD, NANOG and SOX2. Arrowheads indicate morphologically wild-type germ cells and arrows indicate neoplasias. E-cadherin (ECAD; red) labels both germ cells and neoplasias in all frames. The frequency of neoplasia
    Figure Legend Snippet: Early neoplasias express high levels of ECAD, NANOG and SOX2. Arrowheads indicate morphologically wild-type germ cells and arrows indicate neoplasias. E-cadherin (ECAD; red) labels both germ cells and neoplasias in all frames. The frequency of neoplasia

    Techniques Used:

    36) Product Images from "Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide"

    Article Title: Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201507023

    SOD mimetics EUK-8 and EUK-134 restore normal O 2 •− levels and rescue defective NSCs in Fip200 GFAP cKO mice. (A–I) Ctrl and Fip200 GFAP cKO mice at P28 were treated with vehicle, EUK-8, or EUK-134. (A) DHE and DAPI fluorescence in SVZ. Arrows indicate SVZ cells. Dotted lines indicate the boundaries of the SVZ ( n = 4 mice each). (B) Mean ± SEM of SVZ cellularity per section ( n = 3 mice each). (C and D) Mean ± SEM of GFAP + Nestin + (C) and GFAP + SOX2 + (D) cells per section ( n = 4 mice each). (E and F) Mean ± SEM of the percentage of GFAP + Nestin + Ki67 + to total Ki67 + cells (E) or total GFAP + Nestin + cells (F) in SVZ ( n = 4 mice each). (G–I) Mean ± SEM of TUNEL + (G) or DCX + (H) cells per SVZ section and NeuN + cells in the OB per square millimeter (I) of mice ( n = 4 mice each). E, ependymal layer; LV, lateral ventricle; ST, striatum. Bars, 50 µm. *, P
    Figure Legend Snippet: SOD mimetics EUK-8 and EUK-134 restore normal O 2 •− levels and rescue defective NSCs in Fip200 GFAP cKO mice. (A–I) Ctrl and Fip200 GFAP cKO mice at P28 were treated with vehicle, EUK-8, or EUK-134. (A) DHE and DAPI fluorescence in SVZ. Arrows indicate SVZ cells. Dotted lines indicate the boundaries of the SVZ ( n = 4 mice each). (B) Mean ± SEM of SVZ cellularity per section ( n = 3 mice each). (C and D) Mean ± SEM of GFAP + Nestin + (C) and GFAP + SOX2 + (D) cells per section ( n = 4 mice each). (E and F) Mean ± SEM of the percentage of GFAP + Nestin + Ki67 + to total Ki67 + cells (E) or total GFAP + Nestin + cells (F) in SVZ ( n = 4 mice each). (G–I) Mean ± SEM of TUNEL + (G) or DCX + (H) cells per SVZ section and NeuN + cells in the OB per square millimeter (I) of mice ( n = 4 mice each). E, ependymal layer; LV, lateral ventricle; ST, striatum. Bars, 50 µm. *, P

    Techniques Used: Mouse Assay, Fluorescence, TUNEL Assay

    Postnatal NSC pool and neurogenesis are intact in Atg5 GFAP cKO and Atg16L1 GFAP cKO mice. (A and B) H E staining of SVZ (A) and DG (B) from Ctrl, Atg5 GFAP cKO, and Atg16L1 GFAP cKO mice at P28. Dotted lines indicate the SVZ and DG boundaries. SVZ cellularity (mean ± SEM; A) and DG area (B) per section from different mice ( n = 4 mice each). (C and D) Immunofluorescence for GFAP, Nestin (C), SOX2 (D), and DAPI in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ at P28. Mean ± SEM of GFAP+ and Nestin+ (C) and GFAP+ and Sox2+ (D) cell number per SVZ section ( n = 3 mice each). (E and F) Immunofluorescence for Ki67 and DAPI (E) and TUNEL and DAPI (F) in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ and RMS at P28. Arrows mark examples of TUNEL+ cells in insets. Mean ± SEM of the Ki67+ (E) and TUNEL+ (F) cell number per SVZ section ( n = 3 mice each). (G) Immunofluorescence for DCX and DAPI in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ and RMS at P28. Mean ± SEM of DCX+ cell number per SVZ section ( n = 3 mice each). (H) Mean ± SEM of the number and size of primary and secondary neurospheres from Ctrl, Atg5 GFAP cKO and Atg16L1 GFAP cKO mice at P28 ( n = 3 mice each). CC, corpus callosum; E, ependymal layer; LV, lateral ventricle; ST, striatum. Bars: (A and B) 100 µm; (C–G and insets) 40 µm. *, P
    Figure Legend Snippet: Postnatal NSC pool and neurogenesis are intact in Atg5 GFAP cKO and Atg16L1 GFAP cKO mice. (A and B) H E staining of SVZ (A) and DG (B) from Ctrl, Atg5 GFAP cKO, and Atg16L1 GFAP cKO mice at P28. Dotted lines indicate the SVZ and DG boundaries. SVZ cellularity (mean ± SEM; A) and DG area (B) per section from different mice ( n = 4 mice each). (C and D) Immunofluorescence for GFAP, Nestin (C), SOX2 (D), and DAPI in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ at P28. Mean ± SEM of GFAP+ and Nestin+ (C) and GFAP+ and Sox2+ (D) cell number per SVZ section ( n = 3 mice each). (E and F) Immunofluorescence for Ki67 and DAPI (E) and TUNEL and DAPI (F) in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ and RMS at P28. Arrows mark examples of TUNEL+ cells in insets. Mean ± SEM of the Ki67+ (E) and TUNEL+ (F) cell number per SVZ section ( n = 3 mice each). (G) Immunofluorescence for DCX and DAPI in Ctrl, Fip200 GFAP cKO, Atg5 GFAP cKO, and Atg16L1 GFAP cKO SVZ and RMS at P28. Mean ± SEM of DCX+ cell number per SVZ section ( n = 3 mice each). (H) Mean ± SEM of the number and size of primary and secondary neurospheres from Ctrl, Atg5 GFAP cKO and Atg16L1 GFAP cKO mice at P28 ( n = 3 mice each). CC, corpus callosum; E, ependymal layer; LV, lateral ventricle; ST, striatum. Bars: (A and B) 100 µm; (C–G and insets) 40 µm. *, P

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, TUNEL Assay

    p62 KO rescues the maintenance and differentiation defects of Fip200-null NSCs in vivo. (A) H E staining of SVZ and hippocampus from FIP GFAP cKO and 2cKO mice at P28. Dotted lines indicate boundaries of SVZ and DG ( n = 4 mice each). (B) Mean ± SEM of SVZ cellularity (left) and DG area (right) per section from Ctrl, FIP GFAP cKO, 2cKO, and p62 KO mice at P28. (C and D) Immunofluorescence for GFAP, Nestin (C), SOX2 (D), and DAPI in SVZ from Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO mice at P28. Mean ± SEM of GFAP+ and Nestin+ (C) and GFAP+ and Sox2+ (D) cell number per SVZ section ( n = 4 mice each). (E) Immunofluorescence of long-term retained BrdU and DAPI in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of BrdU+ cell number per SVZ section ( n = 4 mice each). (F and G) Immunofluorescence for p62 and DAPI (F; n = 3 mice each) and TUNEL and DAPI (G) in SVZ of Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO mice at P28. Mean ± SEM of TUNEL+ cells per 100 SVZ cells (G, n = 4 mice each, > 1,000 cells per mouse). (H and I) Immunofluorescence of Nestin, GFAP, and short-term labeled BrdU in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of the percentage of GFAP + Nestin + BrdU + to total GFAP + Nestin + cells (H) or total BrdU + cells (I) per section ( n = 5 mice each). (J and K) Immunofluorescence for Nestin, GFAP, and Ki67 in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of the percentage of GFAP + Nestin + Ki67 + to total GFAP + Nestin + cells (J) or total Ki67 + cells (K) per section ( n = 5 mice each). (L and M) Immunofluorescence for DCX in SVZ (L) and NeuN in olfactory bulb (M) in Ctrl, Fip200 GFAP cKO, 2cKO and p62 KO mice at P28. Mean ± SEM of DCX + cells per section (L) and NeuN + cells per square millimeter (M; n = 4 mice each). Dotted lines indicate the boundaries of SVZ (C–G). CC, corpus callosum; E, ependymal layer; LV, lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream; ST, striatum. Bars: (A, E, and G and insets) 50 µm; (C, D, and insets) 25 µm; (F and insets) 15 µm. *, P
    Figure Legend Snippet: p62 KO rescues the maintenance and differentiation defects of Fip200-null NSCs in vivo. (A) H E staining of SVZ and hippocampus from FIP GFAP cKO and 2cKO mice at P28. Dotted lines indicate boundaries of SVZ and DG ( n = 4 mice each). (B) Mean ± SEM of SVZ cellularity (left) and DG area (right) per section from Ctrl, FIP GFAP cKO, 2cKO, and p62 KO mice at P28. (C and D) Immunofluorescence for GFAP, Nestin (C), SOX2 (D), and DAPI in SVZ from Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO mice at P28. Mean ± SEM of GFAP+ and Nestin+ (C) and GFAP+ and Sox2+ (D) cell number per SVZ section ( n = 4 mice each). (E) Immunofluorescence of long-term retained BrdU and DAPI in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of BrdU+ cell number per SVZ section ( n = 4 mice each). (F and G) Immunofluorescence for p62 and DAPI (F; n = 3 mice each) and TUNEL and DAPI (G) in SVZ of Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO mice at P28. Mean ± SEM of TUNEL+ cells per 100 SVZ cells (G, n = 4 mice each, > 1,000 cells per mouse). (H and I) Immunofluorescence of Nestin, GFAP, and short-term labeled BrdU in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of the percentage of GFAP + Nestin + BrdU + to total GFAP + Nestin + cells (H) or total BrdU + cells (I) per section ( n = 5 mice each). (J and K) Immunofluorescence for Nestin, GFAP, and Ki67 in Ctrl, Fip200 GFAP cKO, 2cKO, and p62 KO SVZ at P28. Mean ± SEM of the percentage of GFAP + Nestin + Ki67 + to total GFAP + Nestin + cells (J) or total Ki67 + cells (K) per section ( n = 5 mice each). (L and M) Immunofluorescence for DCX in SVZ (L) and NeuN in olfactory bulb (M) in Ctrl, Fip200 GFAP cKO, 2cKO and p62 KO mice at P28. Mean ± SEM of DCX + cells per section (L) and NeuN + cells per square millimeter (M; n = 4 mice each). Dotted lines indicate the boundaries of SVZ (C–G). CC, corpus callosum; E, ependymal layer; LV, lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream; ST, striatum. Bars: (A, E, and G and insets) 50 µm; (C, D, and insets) 25 µm; (F and insets) 15 µm. *, P

    Techniques Used: In Vivo, Staining, Mouse Assay, Immunofluorescence, TUNEL Assay, Labeling

    37) Product Images from "The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice"

    Article Title: The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice

    Journal: eLife

    doi: 10.7554/eLife.18197

    Knockdown of TBC1D3 in human vRGs inhibits the generation of oRGs. ( A ) Detection of TBC1D3 protein levels in Hela cells transfected with pSuper-siTBC1D3 plasmids, with a scramble sequence as the control. ( B ) Paradigm of culture and electroporation of human brain slice. ( C – E ) The VZ of human brain slices at GW14.5 ( C ), GW17.1 ( D ), GW13.5 ( E ) were transfected with pSuper-siTBC1D3 plasmids or a plasmid encoding scrambled sequence as the control, without or with co-transfection with TBC1D3 expression plasmid (pCS2-Myc-TBC1D3) by electroporation method as described in ( B ), followed by staining with Sox2 antibody at 72 hr post electroporation. Scale bars, 50 μm. ( F ) Quantification for the percentage of Sox2 + cells among total EGFP + cells in basal regions (control: n = 4 slices, mean = 55.91, SEM = 5.76; siTBC1D3: n = 4 slices, mean = 13.32, SEM = 1.46; siTBC1D3 plus TBC1D3: n = 3 slices, mean = 37.47, SEM = 3.12). p = 0.0002, control vs siTBC1D3; p = 0.004, siTBC1D3 vs siTBC1D3 plus TBC1D3. ( G ) Human ReNeuron cells were transfected with siTBC1D3 or control plasmid for 3 days followed by treatment with actinomycin D for 4 hr. The mRNA levels of Cdh2 in ReNeuron cells after actinomycin D treatment were quantified (control: n = 6 experiments; mean = 66.99, SEM = 6.21; siTBC1D3: mean = 96.62, SEM = 7.62; p = 0.003), normalized to that in cells with 0 hr of actinomycin D treatment. DOI: http://dx.doi.org/10.7554/eLife.18197.016
    Figure Legend Snippet: Knockdown of TBC1D3 in human vRGs inhibits the generation of oRGs. ( A ) Detection of TBC1D3 protein levels in Hela cells transfected with pSuper-siTBC1D3 plasmids, with a scramble sequence as the control. ( B ) Paradigm of culture and electroporation of human brain slice. ( C – E ) The VZ of human brain slices at GW14.5 ( C ), GW17.1 ( D ), GW13.5 ( E ) were transfected with pSuper-siTBC1D3 plasmids or a plasmid encoding scrambled sequence as the control, without or with co-transfection with TBC1D3 expression plasmid (pCS2-Myc-TBC1D3) by electroporation method as described in ( B ), followed by staining with Sox2 antibody at 72 hr post electroporation. Scale bars, 50 μm. ( F ) Quantification for the percentage of Sox2 + cells among total EGFP + cells in basal regions (control: n = 4 slices, mean = 55.91, SEM = 5.76; siTBC1D3: n = 4 slices, mean = 13.32, SEM = 1.46; siTBC1D3 plus TBC1D3: n = 3 slices, mean = 37.47, SEM = 3.12). p = 0.0002, control vs siTBC1D3; p = 0.004, siTBC1D3 vs siTBC1D3 plus TBC1D3. ( G ) Human ReNeuron cells were transfected with siTBC1D3 or control plasmid for 3 days followed by treatment with actinomycin D for 4 hr. The mRNA levels of Cdh2 in ReNeuron cells after actinomycin D treatment were quantified (control: n = 6 experiments; mean = 66.99, SEM = 6.21; siTBC1D3: mean = 96.62, SEM = 7.62; p = 0.003), normalized to that in cells with 0 hr of actinomycin D treatment. DOI: http://dx.doi.org/10.7554/eLife.18197.016

    Techniques Used: Transfection, Sequencing, Electroporation, Slice Preparation, Plasmid Preparation, Cotransfection, Expressing, Staining

    Cortical basal progenitors are increased in the cortex of TBC1D3-transgenic mice. ( A and B ) Immunostaining for PH3 in WT and TBC1D3 TG mice at E12.5. White dash lines in enlarged areas ( B ) indicate cortical surfaces, and yellow dash lines indicate the boundary between apical and basal regions. Scale bars, 50 μm. ( C and D ) Quantification for the density of PH3 + cells distributed radially ( C , from ventricular to pial surface) or in apical/basal regions ( D ), respectively. Apical: n = 3 mice, mean = 453.37, SEM = 24.89 for WT, n = 4 mice, mean = 432.36, SEM = 24.99 for TG; Basal: mean = 251.14, SEM = 13.09 for WT; mean = 378.62, SEM = 42.49 for TG. p-values are 0.570 (apical) and 0.017 (basal). ( E ) Staining for Pax6, Sox2, and Tbr2 in E12.5 WT and TG mice. Scale bar, 50 μm. ( F ) The densitys of Pax6 + cells in the cortex of WT and TG mice were quantified (WT: n = 3 mice, mean = 55.5, SEM = 1.67; TG: n = 4 mice, mean = 61.59, SEM = 1.43). p = 0.014. DOI: http://dx.doi.org/10.7554/eLife.18197.023
    Figure Legend Snippet: Cortical basal progenitors are increased in the cortex of TBC1D3-transgenic mice. ( A and B ) Immunostaining for PH3 in WT and TBC1D3 TG mice at E12.5. White dash lines in enlarged areas ( B ) indicate cortical surfaces, and yellow dash lines indicate the boundary between apical and basal regions. Scale bars, 50 μm. ( C and D ) Quantification for the density of PH3 + cells distributed radially ( C , from ventricular to pial surface) or in apical/basal regions ( D ), respectively. Apical: n = 3 mice, mean = 453.37, SEM = 24.89 for WT, n = 4 mice, mean = 432.36, SEM = 24.99 for TG; Basal: mean = 251.14, SEM = 13.09 for WT; mean = 378.62, SEM = 42.49 for TG. p-values are 0.570 (apical) and 0.017 (basal). ( E ) Staining for Pax6, Sox2, and Tbr2 in E12.5 WT and TG mice. Scale bar, 50 μm. ( F ) The densitys of Pax6 + cells in the cortex of WT and TG mice were quantified (WT: n = 3 mice, mean = 55.5, SEM = 1.67; TG: n = 4 mice, mean = 61.59, SEM = 1.43). p = 0.014. DOI: http://dx.doi.org/10.7554/eLife.18197.023

    Techniques Used: Transgenic Assay, Mouse Assay, Immunostaining, Staining

    oRG-like cells and IPs increase in the basal region of TBC1D3-expressing mouse cortex. ( A and C ) Staining for phospho-Vimention (p-Vim) and Sox2 ( A ) or p-Vim and Pax6 ( C ) in E15.5 mice subjected to IUE at E13.5. Dash lines indicate the boundary between basal (outer VZ) and apical (VZ) regions in mouse neocortex. Note the cells double positive for p-Vim and Sox2 or Pax6 (yellow arrows) with the basal process with coherent patterned intermittent p-Vim signals. Scale bars, 50 μm. ( B and D ) Quantification for the number of p-Vim + Sox2 + (control: n = 6 mice, mean = 6.79, SEM = 1.02; TBC1D3: n = 9 mice, mean = 22.53, SEM = 2.91; p = 0.0009) or p-Vim + Pax6 + (control: n = 15 mice, mean = 3.10, SEM = 1.16; TBC1D3: n = 19 mice, mean = 8.63, SEM = 1.39; p = 0.009) cells with basal processes in the basal region of electroporated cortex per unit length along the VZ surface. ( E ) Tbr2 staining for E17.5 mouse brains, which were subjected to IUE at E13.5 with TBC1D3 or vehicle control, together with YFP. Dash lines indicate boundaries between apical (VZ) and basal (outer VZ) regions. Scale bar, 50 μm. ( F ) Quantification for the percentage of Tbr2 + cells among electroporated YFP + cells in apical (control: n = 4 mice, mean = 12.40, SEM = 0.79; TBC1D3: n= 7 mice, mean = 12.61, SEM = 2.01; p = 0.286) and basal regions (control: mean = 1.73, SEM = 0.49; TBC1D3: mean = 6.45, SEM = 0.72; p = 0.006). DOI: http://dx.doi.org/10.7554/eLife.18197.012
    Figure Legend Snippet: oRG-like cells and IPs increase in the basal region of TBC1D3-expressing mouse cortex. ( A and C ) Staining for phospho-Vimention (p-Vim) and Sox2 ( A ) or p-Vim and Pax6 ( C ) in E15.5 mice subjected to IUE at E13.5. Dash lines indicate the boundary between basal (outer VZ) and apical (VZ) regions in mouse neocortex. Note the cells double positive for p-Vim and Sox2 or Pax6 (yellow arrows) with the basal process with coherent patterned intermittent p-Vim signals. Scale bars, 50 μm. ( B and D ) Quantification for the number of p-Vim + Sox2 + (control: n = 6 mice, mean = 6.79, SEM = 1.02; TBC1D3: n = 9 mice, mean = 22.53, SEM = 2.91; p = 0.0009) or p-Vim + Pax6 + (control: n = 15 mice, mean = 3.10, SEM = 1.16; TBC1D3: n = 19 mice, mean = 8.63, SEM = 1.39; p = 0.009) cells with basal processes in the basal region of electroporated cortex per unit length along the VZ surface. ( E ) Tbr2 staining for E17.5 mouse brains, which were subjected to IUE at E13.5 with TBC1D3 or vehicle control, together with YFP. Dash lines indicate boundaries between apical (VZ) and basal (outer VZ) regions. Scale bar, 50 μm. ( F ) Quantification for the percentage of Tbr2 + cells among electroporated YFP + cells in apical (control: n = 4 mice, mean = 12.40, SEM = 0.79; TBC1D3: n= 7 mice, mean = 12.61, SEM = 2.01; p = 0.286) and basal regions (control: mean = 1.73, SEM = 0.49; TBC1D3: mean = 6.45, SEM = 0.72; p = 0.006). DOI: http://dx.doi.org/10.7554/eLife.18197.012

    Techniques Used: Expressing, Staining, Mouse Assay

    38) Product Images from "Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector"

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00415

    A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .
    Figure Legend Snippet: A long-lasting NSC population in the adult hippocampus. GFP-positive neuroblasts were observed in the SGZ 6 months after LV PGK-GFP injection. Some GFP + cells incorporated BrdU (A) and maintained the expression of SOX2 (B) , indicating that LV PGK-GFP-labeled NSC populations retained the proliferation capacity over a six-month tracing period. Many of the GFP-labeled cells expressed the early neuronal marker doublecortin (DCX; C ). Higher magnification picture of the triple-labeled cells GFP/DCX/BrdU (D) .

    Techniques Used: Injection, Expressing, Labeling, Marker

    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.
    Figure Legend Snippet: Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Techniques Used: Injection, Expressing, Labeling, Staining

    Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .
    Figure Legend Snippet: Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .

    Techniques Used: Labeling, In Vivo, In Vitro, Derivative Assay

    39) Product Images from "B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium"

    Article Title: B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

    Journal: Chinese Journal of Cancer

    doi: 10.5732/cjc.012.10228

    Stem cell marker expression in U251 cells. A, cells in tumor spheres all expressed neural precursor cell markers nestin (green, left panel) and SOX2 (red, middle panel). B, CD133-positive cells (green, left panel) exist both in tumor spheres and in migrating U251 cells with expression of SOX2 (red, middle panel). All right panels show merge images.
    Figure Legend Snippet: Stem cell marker expression in U251 cells. A, cells in tumor spheres all expressed neural precursor cell markers nestin (green, left panel) and SOX2 (red, middle panel). B, CD133-positive cells (green, left panel) exist both in tumor spheres and in migrating U251 cells with expression of SOX2 (red, middle panel). All right panels show merge images.

    Techniques Used: Marker, Expressing

    Characteristics of U251 cells cultured in different mediums. A, all cells expressed the neural precursor cell markers nestin (green, first panel) and SOX2 (red, second panel) when tumor spheres were seeded on pretreated coverslips. B, cells that migrated out from tumor spheres cultured in serum-free medium were all nestin-positive (green, first panel), and a little cells coexpressed GFAP (red, second panel). C, when tumor spheres were cultured in serum-containing medium for 48 h, nestin was undetectable (green, first panel) but GFAP was highly expressed (red, second, panel). DAPI-counterstained nuclei are shown in blue. All right panels show merge images.
    Figure Legend Snippet: Characteristics of U251 cells cultured in different mediums. A, all cells expressed the neural precursor cell markers nestin (green, first panel) and SOX2 (red, second panel) when tumor spheres were seeded on pretreated coverslips. B, cells that migrated out from tumor spheres cultured in serum-free medium were all nestin-positive (green, first panel), and a little cells coexpressed GFAP (red, second panel). C, when tumor spheres were cultured in serum-containing medium for 48 h, nestin was undetectable (green, first panel) but GFAP was highly expressed (red, second, panel). DAPI-counterstained nuclei are shown in blue. All right panels show merge images.

    Techniques Used: Cell Culture

    40) Product Images from "Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity"

    Article Title: Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.09.019

    CD44+/CD24+ antigens identify TRTICs and GSCs. a. Flow cytometry plot showing the expression of CD24 and CD44 in OSU53GSC and PKAC2GSC cells after 10 Gy radiation treatment (n = 3). b. Phase contrast and GFP+/CD24+/CD44+ GFP cells showing sphere-forming ability at day1 to day15. Microscopic images of OSU53GSC and OSU68GSC CD44+/CD24+ and CD44-/CD24- TRTICs 10 days after sorting pure population (n = 3). c. Kaplan-Meier survival plots of NOD-SCID mice bearing tumors from CD24+/CD44+ and CD24-/CD44- populations of OSU53GSC and OSU68GSC cells (n = 3) (log-rank (Mantel-Cox) test). d. Immunohistochemistry of Sox2, Nestin, CD44, Hif-1α and VEGF in U87 TRTIC-derived tumors (n = 3). The Hematoxylin and Eosin stained tumor section demonstrates the adjacent normal tissue in the Sox2, Nestin and CD44 images. The Hif-1α and VEGF adjacent normal tissue is represented as individual boxes to the left of their respective tumor staining. e. Western blots showing the protein expression pattern of unsorted, CD44+/CD24+ and CD24-/CD44- OSU53GSC and OSU68GSC cells (n = 3). Scale bar = 50 μm.
    Figure Legend Snippet: CD44+/CD24+ antigens identify TRTICs and GSCs. a. Flow cytometry plot showing the expression of CD24 and CD44 in OSU53GSC and PKAC2GSC cells after 10 Gy radiation treatment (n = 3). b. Phase contrast and GFP+/CD24+/CD44+ GFP cells showing sphere-forming ability at day1 to day15. Microscopic images of OSU53GSC and OSU68GSC CD44+/CD24+ and CD44-/CD24- TRTICs 10 days after sorting pure population (n = 3). c. Kaplan-Meier survival plots of NOD-SCID mice bearing tumors from CD24+/CD44+ and CD24-/CD44- populations of OSU53GSC and OSU68GSC cells (n = 3) (log-rank (Mantel-Cox) test). d. Immunohistochemistry of Sox2, Nestin, CD44, Hif-1α and VEGF in U87 TRTIC-derived tumors (n = 3). The Hematoxylin and Eosin stained tumor section demonstrates the adjacent normal tissue in the Sox2, Nestin and CD44 images. The Hif-1α and VEGF adjacent normal tissue is represented as individual boxes to the left of their respective tumor staining. e. Western blots showing the protein expression pattern of unsorted, CD44+/CD24+ and CD24-/CD44- OSU53GSC and OSU68GSC cells (n = 3). Scale bar = 50 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Immunohistochemistry, Derivative Assay, Staining, Western Blot

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    Article Snippet: .. Incubation was performed at 4°C overnight with primary antibodies at the following concentrations: goat anti-brachyury 1 µg ml−1 (AF2085, R & D), rabbit anti-Sox2 5 µg m−1 (ab5603, Millipore), rabbit anti-β-III-tubulin 1 µg ml−1 (T2200, Sigma-Aldrich), mouse anti-HB9 1.75 µg ml−1 (81.5C10, Developmental Studies Hybridoma Bank) and rabbit anti-islet 1 2.5 µg ml−1 (ab20670, Abcam). .. Fluorochrome-conjugated secondary antibodies used were the following: anti-goat Alexa647-conjugated 4 µg ml−1 (A21447, Invitrogen), anti-rabbit Alexa488-conjugated 4 µg ml−1 (A21206, Molecular Probes) and anti-mouse Alexa594-conjugated 4 µg ml−1 (A11032, Molecular Probes).

    Article Title: The TGF-β System As a Potential Pathogenic Player in Disease Modulation of Amyotrophic Lateral Sclerosis
    Article Snippet: .. Afterward, the membranes were blocked with 5% BSA (Albumin-IgG-free, Roth, Karlsruhe, Germany), diluted with TBS-T for 1 h at RT, the primary antibodies (diluted in 0.5% BSA in TBS-T) were added and incubated at 4°C for 2 days [rabbit anti-TGF-β1 (1:300; Acris), rabbit anti-TGF-β2 (1:500; BioVision), rabbit anti-TGF-βRI (1:1,000; Abcam), rabbit anti-TGF-βRII (1:1,000; Aviva), rabbit anti-Nestin (1:100; Abcam), rabbit anti-SOX-2 (1:750; Millipore), rabbit anti-MSI1 (1:2,000; Abcam), rabbit anti-DCX (1:1,000; Cellsignaling), rabbit anti-Fibronectin (1:250; ProteinTech), and rabbit anti-CollagenIV (1:1,000; Abcam)]. ..

    Immunostaining:

    Article Title: Oct-3/4 Maintains the Proliferative Embryonic Stem Cell State via Specific Binding to a Variant Octamer Sequence in the Regulatory Region of the UTF1 Locus
    Article Snippet: .. Immunostaining was performed using an anti-Sox-2 (Chemicon) or anti-cytokeratin 7 (Chemicon) antibody together with appropriate Alexa-Fluor dye-conjugated secondary antibodies (Molecular Probes) as described by Miyagi et al. ( ). ..

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  • 99
    Millipore anti sox2
    Tamoxifen induces <t>SOX2</t> to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
    Anti Sox2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti sox2 - by Bioz Stars, 2020-07
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    94
    Millipore sox2
    Phenotype of spinal cord-derived LeX+ cells. a , LeX+ cells were isolated from VZ cells of E10.5 mouse spinal cords by immunomagnetic selection and expanded in vitro . b , FACS analysis of negative control cells. c , FACS analysis showed that positive fractions were 70–98% pure for LeX+ cells. d–f , Isolated LeX+ cells formed a monolayer of cells in culture that were positive for both nestin ( e , red) and LeX ( d , green). g–i , Phase-contrast and immunofluorescence images of LeX+ cells expressing both <t>SOX2</t> (red) and the proliferation marker PCNA (light blue). j , k , LeX+ cells coexpressed the stem cell markers SOX 2 (green) and Musashi-1 (red). Nuclei are labeled with DAPI (4′,6-diamidino-2-phenylindole; blue). f , i , and l show the merged images. Scale bar: d–f , 40 μm; g–l , 50 μm.
    Sox2, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 252 article reviews
    Price from $9.99 to $1999.99
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    Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Journal: Cancers

    Article Title: TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer

    doi: 10.3390/cancers11020210

    Figure Lengend Snippet: Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Article Snippet: The primary antibodies used were anti-SOX2 (Millipore, MA, USA; cat. AB5603, 1:50) anti-TARBP2 (Thermo, MA, USA; cat. LF-MA0209, Clone 46D1, 1:600) for 30 min.

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Western Blot, Transfection, shRNA, MTT Assay, Blocking Assay

    Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.

    Journal: Cancers

    Article Title: TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer

    doi: 10.3390/cancers11020210

    Figure Lengend Snippet: Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.

    Article Snippet: The primary antibodies used were anti-SOX2 (Millipore, MA, USA; cat. AB5603, 1:50) anti-TARBP2 (Thermo, MA, USA; cat. LF-MA0209, Clone 46D1, 1:600) for 30 min.

    Techniques: Expressing, Immunohistochemistry

    Expression analysis of molecular markers of iN cells. A–H , mCherry + cells in the dorsal midbrain derived from WT mice that were infected with the virus AAV–Ascl1/mCherry at P12–P15 were collected by FACS on day 4 (D4, black bars), day 10 (D10, red bars), and day 30 (D30, blue bars) after infection. The expression of the astrocyte markers Gfap ( A ), S100 β ( B ), and Acsbg1 ( C ), the neuronal markers Tuj1 ( D ), Map2 ( E ), and NeuN ( F ), the neural progenitor markers Sox2 ( G ) and Pax6 ( H ), and the midbrain neural progenitor markers En1 , En2 , Pax3 , and Pax7 ( I ) was examined by qRT-PCR. The neurospheres (yellow bars) derived from the SVZ of mice at P0 were used as positive controls for detecting the expression of Sox2 and Pax6 . The cells derived from E12.5 midbrain (purple bars) were used as positive controls for detecting the expression of En1 , En2 , Pax3 , and Pax7 .

    Journal: The Journal of Neuroscience

    Article Title: Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo

    doi: 10.1523/JNEUROSCI.3975-14.2015

    Figure Lengend Snippet: Expression analysis of molecular markers of iN cells. A–H , mCherry + cells in the dorsal midbrain derived from WT mice that were infected with the virus AAV–Ascl1/mCherry at P12–P15 were collected by FACS on day 4 (D4, black bars), day 10 (D10, red bars), and day 30 (D30, blue bars) after infection. The expression of the astrocyte markers Gfap ( A ), S100 β ( B ), and Acsbg1 ( C ), the neuronal markers Tuj1 ( D ), Map2 ( E ), and NeuN ( F ), the neural progenitor markers Sox2 ( G ) and Pax6 ( H ), and the midbrain neural progenitor markers En1 , En2 , Pax3 , and Pax7 ( I ) was examined by qRT-PCR. The neurospheres (yellow bars) derived from the SVZ of mice at P0 were used as positive controls for detecting the expression of Sox2 and Pax6 . The cells derived from E12.5 midbrain (purple bars) were used as positive controls for detecting the expression of En1 , En2 , Pax3 , and Pax7 .

    Article Snippet: Primary antibodies were as follows: mouse anti-GFAP (1:1000; MAB360; Millipore), rabbit GFAP (1:1000; Z0334; DAKO), mouse anti-Tuj1 (1:500; MMS-435P; Covance), mouse anti-Map2 (1:500; M4403; Sigma), rabbit anti-GFP (1:1000; A6455; Invitrogen), chicken anti-GFP (A10262; 1:1000; Invitrogen), mouse anti-NeuN (1:100; MAB377; Millipore), rabbit anti-synapsin I (AB1543; 1:1000; Millipore), rabbit anti-GABA (A2052; 1:3000; Sigma), mouse anti-GAD67 (MAB5406; 1:200; Millipore), guinea pig anti-vesicular GABA transporter (VGAT; 1:200; 131004; Synaptic Systems), rabbit anti-Discosoma red (DsRed; 1:500; 632496; Clontech), mouse anti-DsRed (1:100; sc-81595; Santa Cruz Biotechnology), guinea pig anti-VGLUT2 (1:400; VGluT2-GP-Af810; Frontier Institute), rabbit anti-acyl-CoA synthetase bubblegum family member 1 (Acsbg1; 1:100; ab65154; Abcam), rabbit anti-Sox2 (1:500; AB5603; Millipore), mouse anti-S100β (1:1000; S2532; Sigma), rabbit anti-excitatory amino acid transporter 1 (1:500; ab416; Abcam), mouse anti-glutamine synthetase (GS; 1:200; 610518; BD Biosciences), rabbit anti-NG2 (1:200; AB5320; Millipore), rabbit anti-ionized calcium-binding adapter molecule 1 (IBAI; 1:500; 019-19741; Wako), mouse anti-CNPase (1:500; ab6319; Abcam), mouse anti-O4 (1:500; MAB345; Millipore), rabbit anti-Olig2 (1:500; AB9610; Millipore), rabbit anti-doublecortin (DCX; 1:500; ab77450; Abcam), mouse anti-Ascl1 (1:200; 556604; BD Biosciences), rabbit anti-Ki67 (1:200; RM-9106; Thermo Fisher Scientific), mouse anti-BrdU (1:200; B2531; Sigma), and mouse anti-GST-π (1:50; 610718; BD Biosciences).

    Techniques: Expressing, Derivative Assay, Mouse Assay, Infection, FACS, Quantitative RT-PCR

    Evidence for MIR450 cluster as a potential repressor of SOX2. (A): Schematic representation of the 3′UTR of human SOX2 and predicted binding site of miR‐450a and miR‐450b identified by TargetScan. (B): Sequence and complementation of the predicted binding site. (C): Illustration of the human MIR450 cluster (defined by miRBase) that includes six miRNAs genes. (D): Human embryonic stem cells were seeded on collagen IV‐coated dishes in the present of corneal fibroblast conditional media to induce corneal epithelial differentiation for the indicated time. Relative expression of the indicated miRNAs is shown and data represent the normalized expression as fold change in expression relative to undifferentiated cells. (E): Wholemount in situ hybridization for miR‐450b on mouse embryos of the indicated embryonic day. Increased magnifications are shown from left to right, and lens is annotated by white arrowheads. (F): Immunofluorescence staining of SOX2 on mouse head sections at E10.5 and E11.5. Nuclei were counterstained with DAPI. (G, H): In situ hybridization of miR‐450b on whole cornea (G) or sections of cornea (H) of 2‐month‐old mice. Scale bars are 250 μm (E, G) and 25 μm (F, H) . Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; lp, lens pit; lv, lens vesicle; oc, optic cup; pce, presumptive corneal epithelium; UTR, untranslated region.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: Evidence for MIR450 cluster as a potential repressor of SOX2. (A): Schematic representation of the 3′UTR of human SOX2 and predicted binding site of miR‐450a and miR‐450b identified by TargetScan. (B): Sequence and complementation of the predicted binding site. (C): Illustration of the human MIR450 cluster (defined by miRBase) that includes six miRNAs genes. (D): Human embryonic stem cells were seeded on collagen IV‐coated dishes in the present of corneal fibroblast conditional media to induce corneal epithelial differentiation for the indicated time. Relative expression of the indicated miRNAs is shown and data represent the normalized expression as fold change in expression relative to undifferentiated cells. (E): Wholemount in situ hybridization for miR‐450b on mouse embryos of the indicated embryonic day. Increased magnifications are shown from left to right, and lens is annotated by white arrowheads. (F): Immunofluorescence staining of SOX2 on mouse head sections at E10.5 and E11.5. Nuclei were counterstained with DAPI. (G, H): In situ hybridization of miR‐450b on whole cornea (G) or sections of cornea (H) of 2‐month‐old mice. Scale bars are 250 μm (E, G) and 25 μm (F, H) . Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; lp, lens pit; lv, lens vesicle; oc, optic cup; pce, presumptive corneal epithelium; UTR, untranslated region.

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: Binding Assay, Sequencing, Expressing, In Situ Hybridization, Immunofluorescence, Staining, Mouse Assay

    SOX2 regulates long‐term colony‐forming efficiency and cell proliferation. Limbal cells were transfected with siSOX2 or siP63 or siCtl and 48 hours later, subjected to clonogenicity test as detailed in Materials and Methods section. Colonies were visualized by Rhodamin staining 3 weeks later (A, D) , and quantification of the number of colonies relative to control (B, E) and the average size of colonies (C, F) was performed by Nis‐Element software as detailed in Materials and Methods section. (G–I): Limbal stem/progenitor cells were transfected with siSOX2 or siCtl and 72 hours later, cells were immunostained for the proliferative marker Ki67 (G) , and quantification (by Nis‐Element software) of the relative number of Ki67‐positive cells is shown (H) . Transfectents were grown for 72 hours and then subjected to alamar blue viability test (I) . (B, C, E, F, H, I): Data represent mean ± SD, n = 3. Significance assessed by t test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: SOX2 regulates long‐term colony‐forming efficiency and cell proliferation. Limbal cells were transfected with siSOX2 or siP63 or siCtl and 48 hours later, subjected to clonogenicity test as detailed in Materials and Methods section. Colonies were visualized by Rhodamin staining 3 weeks later (A, D) , and quantification of the number of colonies relative to control (B, E) and the average size of colonies (C, F) was performed by Nis‐Element software as detailed in Materials and Methods section. (G–I): Limbal stem/progenitor cells were transfected with siSOX2 or siCtl and 72 hours later, cells were immunostained for the proliferative marker Ki67 (G) , and quantification (by Nis‐Element software) of the relative number of Ki67‐positive cells is shown (H) . Transfectents were grown for 72 hours and then subjected to alamar blue viability test (I) . (B, C, E, F, H, I): Data represent mean ± SD, n = 3. Significance assessed by t test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: Transfection, Staining, Software, Marker

    P63 rescues stemness in SOX2 knockdown cells. Limbal cells were co‐transfected with siSOX2 or siCtl and P63 expression plasmid or empty plasmid (veh). Seventy‐two hours later, cells were subjected to clonogenicity test (A) , and number of the colonies were quantified by Nis‐Element software as detailed in Materials and Methods section (B) . (C): Cells were taken for trypan blue assay to quantify dead cells after transfection of indicated factors. Quantitative real‐time polymerase chain reaction analysis of the indicated markers of stem/progenitor cells were performed (D) . (B–D): Data represents mean ± SD, n = 3. Statistical significance was assessed by one‐way analysis of varaince followed by Tukey′s test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: P63 rescues stemness in SOX2 knockdown cells. Limbal cells were co‐transfected with siSOX2 or siCtl and P63 expression plasmid or empty plasmid (veh). Seventy‐two hours later, cells were subjected to clonogenicity test (A) , and number of the colonies were quantified by Nis‐Element software as detailed in Materials and Methods section (B) . (C): Cells were taken for trypan blue assay to quantify dead cells after transfection of indicated factors. Quantitative real‐time polymerase chain reaction analysis of the indicated markers of stem/progenitor cells were performed (D) . (B–D): Data represents mean ± SD, n = 3. Statistical significance was assessed by one‐way analysis of varaince followed by Tukey′s test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

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

    miR‐450b represses SOX2 and induces differentiation of limbal epithelial stem/progenitor cells. (A): 293HEK cells were co‐transfected with SOX2‐3 ′ UTR luciferase plasmid or with a mutated plasmid with disrupted miR‐450a, b binding sites ( Mut‐SOX2‐3 ′ UTR , see Fig. S4 ), and with pre‐miR‐450a (PM450a) or pre‐miR‐450b‐5p (PM450b) or both or control (CtlPM), as indicated. Data represent the normalized luciferase activity relative to control sample. (B): Primary human limbal stem/progenitor cells were induced to differentiate for the indicated time and the expression of the indicated genes was examined by quantitative polymerase chain reaction. (C–J): Primary human limbal stem/progenitor cells were transfected with PM or AM or Ctl and then subjected to differentiation for 4 days and Western blot analysis of the indicated genes (C, G) , or transfectants were allowed to grow for 72 hours and then cell viability was tested by alamar blue assay (D, H) , or transfectants were subjected to clonogenicity test and colonies were revealed by rhodamine staining (E, I) and quantified (F, J) by Nis‐Element software. Data represent mean ± SD, n = 3. (A, B): Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test and (D, F, H, J) t test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: miR‐450b represses SOX2 and induces differentiation of limbal epithelial stem/progenitor cells. (A): 293HEK cells were co‐transfected with SOX2‐3 ′ UTR luciferase plasmid or with a mutated plasmid with disrupted miR‐450a, b binding sites ( Mut‐SOX2‐3 ′ UTR , see Fig. S4 ), and with pre‐miR‐450a (PM450a) or pre‐miR‐450b‐5p (PM450b) or both or control (CtlPM), as indicated. Data represent the normalized luciferase activity relative to control sample. (B): Primary human limbal stem/progenitor cells were induced to differentiate for the indicated time and the expression of the indicated genes was examined by quantitative polymerase chain reaction. (C–J): Primary human limbal stem/progenitor cells were transfected with PM or AM or Ctl and then subjected to differentiation for 4 days and Western blot analysis of the indicated genes (C, G) , or transfectants were allowed to grow for 72 hours and then cell viability was tested by alamar blue assay (D, H) , or transfectants were subjected to clonogenicity test and colonies were revealed by rhodamine staining (E, I) and quantified (F, J) by Nis‐Element software. Data represent mean ± SD, n = 3. (A, B): Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test and (D, F, H, J) t test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: Transfection, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, CTL Assay, Western Blot, Alamar Blue Assay, Staining, Software

    SOX2 is co‐expressed with P63 in stem and progenitor cells of the corneal epithelium in vivo and in vitro. (A): Immunofluorescence staining of the indicated proteins was performed on paraffin sections of the adult mouse cornea. The regions of the limbus, peripheral cornea, and corneal center are shown. (B, C, E, F): Primary human limbal epithelial cells were differentiated for the indicated times, and the expression of the indicated marker was tested by quantitative real‐time polymerase chain reaction (qPCR) (B) or Western blot analysis (C) or immunostaining (E, F) . ERK served as loading control in (C) . (D): A comparative qPCR analysis of SOX2 in the following human cells: primary FE, LS, LE, iPSC, and ESCs. (B, D): Data were normalized to housekeeping gene and is presented (mean ± SD, n = 3) as fold increase compared to control sample. Statistical analysis was performed by one‐way analysis of variance followed by Tukey's test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: SOX2 is co‐expressed with P63 in stem and progenitor cells of the corneal epithelium in vivo and in vitro. (A): Immunofluorescence staining of the indicated proteins was performed on paraffin sections of the adult mouse cornea. The regions of the limbus, peripheral cornea, and corneal center are shown. (B, C, E, F): Primary human limbal epithelial cells were differentiated for the indicated times, and the expression of the indicated marker was tested by quantitative real‐time polymerase chain reaction (qPCR) (B) or Western blot analysis (C) or immunostaining (E, F) . ERK served as loading control in (C) . (D): A comparative qPCR analysis of SOX2 in the following human cells: primary FE, LS, LE, iPSC, and ESCs. (B, D): Data were normalized to housekeeping gene and is presented (mean ± SD, n = 3) as fold increase compared to control sample. Statistical analysis was performed by one‐way analysis of variance followed by Tukey's test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining

    SOX2 can activate P63 enhancer and interact with P63 protein. (A, B): The sequence (A) and location (B) of C38 and C40 enhancers within P63 gene. Consensus binding sites of SOX2 and P63 are highlighted in pink and blue, respectively. (C): Schematic representation of luciferase construct containing C38, C40, C38‐C40, and C38‐C40‐mutated constructs lacking the indicated P63 or SOX2 binding sites. (D): HEK293 cells were co‐transfected with the indicated luciferase construct and with SOX2 or P63 or control empty plasmid (−), as indicated. Luciferase activity represents the relative read that was normalized to Renilla and presented as fold increase compared to control sample (mean ± SD, n = 3). Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: SOX2 can activate P63 enhancer and interact with P63 protein. (A, B): The sequence (A) and location (B) of C38 and C40 enhancers within P63 gene. Consensus binding sites of SOX2 and P63 are highlighted in pink and blue, respectively. (C): Schematic representation of luciferase construct containing C38, C40, C38‐C40, and C38‐C40‐mutated constructs lacking the indicated P63 or SOX2 binding sites. (D): HEK293 cells were co‐transfected with the indicated luciferase construct and with SOX2 or P63 or control empty plasmid (−), as indicated. Luciferase activity represents the relative read that was normalized to Renilla and presented as fold increase compared to control sample (mean ± SD, n = 3). Statistical significance was assessed by one‐way analysis of variance followed by Tukey's test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: Sequencing, Binding Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay

    SOX2 prevents cell differentiation. Primary limbal cells were transfected with siSOX2 or control esiRNA, and 72 hours later, the expression of SOX2 and P63 was examined by real‐time polymerase chain reaction (PCR) (A) or Western blot analysis (B) . ERK served as loading control. Quantitative real‐time PCR analysis of the indicated markers of stem/progenitor cells (C) or markers of differentiated cells (D) , or cells were lysed and subjected to Western blot analysis of the indicated markers (E) (ERK served as loading control) or immunostaining of K3 was followed by flow cytometry analysis (F) . (G): The morphological changes upon siSOX2 repression are shown by bright field microscopy (×20 objective). (A, C, D): Data represent mean ± SD, n = 3 and statistical significance was assessed by t test (*, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

    doi: 10.1002/stem.2959

    Figure Lengend Snippet: SOX2 prevents cell differentiation. Primary limbal cells were transfected with siSOX2 or control esiRNA, and 72 hours later, the expression of SOX2 and P63 was examined by real‐time polymerase chain reaction (PCR) (A) or Western blot analysis (B) . ERK served as loading control. Quantitative real‐time PCR analysis of the indicated markers of stem/progenitor cells (C) or markers of differentiated cells (D) , or cells were lysed and subjected to Western blot analysis of the indicated markers (E) (ERK served as loading control) or immunostaining of K3 was followed by flow cytometry analysis (F) . (G): The morphological changes upon siSOX2 repression are shown by bright field microscopy (×20 objective). (A, C, D): Data represent mean ± SD, n = 3 and statistical significance was assessed by t test (*, p

    Article Snippet: The membranes were blocked with trisma base buffer supplemented with 0.1% tween 20 (TBST, Sigma, USA) containing 5% milk (Bio‐Rad, USA) and probed with one of the following antibodies diluted in blocking solution: rabbit anti‐SOX2 (1:1,000, Millipore, USA), mouse anti‐P63 (1:500, 4A4 Santa Cruz Biotechnology, USA), mouse anti‐K14 (1:1,000, Millipore, USA), mouse anti‐K3 (1:1,000, Millipore, USA), goat anti‐K12 (1:1,000, Santa Cruz Biotechnology, USA), and rabbit anti‐ERK (1:3,500, Santa Cruz Biotechnology, USA) at 4°C, overnight, followed by three washes with TBST.

    Techniques: Cell Differentiation, Transfection, esiRNA, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Immunostaining, Flow Cytometry, Cytometry, Microscopy

    Phenotype of spinal cord-derived LeX+ cells. a , LeX+ cells were isolated from VZ cells of E10.5 mouse spinal cords by immunomagnetic selection and expanded in vitro . b , FACS analysis of negative control cells. c , FACS analysis showed that positive fractions were 70–98% pure for LeX+ cells. d–f , Isolated LeX+ cells formed a monolayer of cells in culture that were positive for both nestin ( e , red) and LeX ( d , green). g–i , Phase-contrast and immunofluorescence images of LeX+ cells expressing both SOX2 (red) and the proliferation marker PCNA (light blue). j , k , LeX+ cells coexpressed the stem cell markers SOX 2 (green) and Musashi-1 (red). Nuclei are labeled with DAPI (4′,6-diamidino-2-phenylindole; blue). f , i , and l show the merged images. Scale bar: d–f , 40 μm; g–l , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Motoneuron Transplantation Rescues the Phenotype of SMARD1 (Spinal Muscular Atrophy with Respiratory Distress Type 1)

    doi: 10.1523/JNEUROSCI.2734-09.2009

    Figure Lengend Snippet: Phenotype of spinal cord-derived LeX+ cells. a , LeX+ cells were isolated from VZ cells of E10.5 mouse spinal cords by immunomagnetic selection and expanded in vitro . b , FACS analysis of negative control cells. c , FACS analysis showed that positive fractions were 70–98% pure for LeX+ cells. d–f , Isolated LeX+ cells formed a monolayer of cells in culture that were positive for both nestin ( e , red) and LeX ( d , green). g–i , Phase-contrast and immunofluorescence images of LeX+ cells expressing both SOX2 (red) and the proliferation marker PCNA (light blue). j , k , LeX+ cells coexpressed the stem cell markers SOX 2 (green) and Musashi-1 (red). Nuclei are labeled with DAPI (4′,6-diamidino-2-phenylindole; blue). f , i , and l show the merged images. Scale bar: d–f , 40 μm; g–l , 50 μm.

    Article Snippet: The following proteins were evaluated using the diluted antibodies indicated in parentheses: nestin (mouse monoclonal antibody; 1:200; Millipore Bioscience Research Reagents), LeX (mouse antibody; 1:200; BD Biosciences), Sox2 (rabbit antibody; 1:200; Millipore Bioscience Research Reagents), Musashi-1 (rabbit antibody; 1:200; Millipore Bioscience Research Reagents), proliferating cell nuclear antigen (PCNA; mouse monoclonal antibody; 1:200; Millipore Bioscience Research Reagents), nuclear neural-specific antigen (NeuN; mouse monoclonal antibody; 1:100; Millipore Bioscience Research Reagents), anti-PDGFRα (platelet-derived growth factor receptor α) antibody (clone APA5, 1:200; eBioscience), Olig2 (rabbit polyclonal antibody; 1:500; Millipore Bioscience Research Reagents), Irx3 (rabbit polyclonal antibody; 1:100; Santa Cruz Biotechnology), Nkx2.2 (rabbit polyclonal antibody;1:200; Millipore Bioscience Research Reagents), HOXC6 (goat polyclonal antibody; 1:100; Santa Cruz Biotechnology), HOXC8 (mouse antibody; 1:200; Covance), otx2 (rabbit polyclonal antibody; 1:200; Millipore Bioscience Research Reagents), En1 (rabbit polyclonal antibody; 1:200; Millipore Bioscience Research Reagents), HB9 (rabbit antibody; 1:200; Millipore Bioscience Research Reagents), Islet-1 (rabbit antibody; 1:200; Millipore Bioscience Research Reagents), TuJ-1 (mouse monoclonal antibody; 1:200; Millipore Bioscience Research Reagents), phosphorylated neurofilament (NF)-M and NF-H (mouse monoclonal antibody; 1:200; Millipore Bioscience Research Reagents), microtubule-associated protein 2 (MAP2; mouse monoclonal antibody; 1:100; Sigma-Aldrich), anti-choline acetyltransferase (ChAT; rabbit antibody; 1:100; Millipore Bioscience Research Reagents), and GFP (Alexa 488 rabbit polyclonal antibody; 1:400; Molecular Probes).

    Techniques: Derivative Assay, Isolation, Selection, In Vitro, FACS, Negative Control, Immunofluorescence, Expressing, Marker, Labeling