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  • 99
    Millipore ctip2
    Differentiation of glutamatergic and GABAergic neurons from dorsal and ventral progenitors. ( A , B ) Comparison of the expression of TBR1, <t>CTIP2</t> and ISLET1 between the neurons that were developed from dorsal (control group) and ventral (DKK1 plus SHH group)
    Ctip2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam ctip 2
    Nrg1 expression in cingulate cortex–BLA circuit: heterozygous deletion of type III Nrg1 alters properties of miniature EPSCs in BLA pyramidal neurons. A , Representative confocal image of Nrg-1 (red) and <t>CTIP-2</t> expression in the cingulate cortex.
    Ctip 2, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti ctip2
    Paxillin deficiency in post-mitotic neurons disrupts neuronal positioning. (A) Representative sections of littermate control ( Pxn F/F ) and mutant ( NEX-Cre:Pxn F/F ), with a conditional deletion of paxillin in post-mitotic immature neurons, were immunostained for Cux1 (green). More Cux1 + neurons were found in ectopic deep positions in the mutant cortex (arrow). (B) Box-and-whisker plot distribution of positions of Cux1 + neurons. The mutant showed a broader distribution compared with littermate controls. The mean position of Cux1 + neurons was significantly deeper in the mutant cortex ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ). (C) Representative images of Tle4 + (red) and <t>Ctip2</t> + (green) neurons. (D) Distribution of Tbr1 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ), Tle4 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) and Ctip2 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) neurons. There was no difference in the mean positions of the Tbr1 + , Tle4 + and Ctip2 + neurons between the genotypes. *** P
    Anti Ctip2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam ctip2 antibody
    In vivo differentiation of human and chimpanzee neurons post-transplantation. ( A ) Images of pyramidal neurons (arrowheads) transplanted in mouse cortex showing co-localization with deep layer (layer V-VI) cortical marker <t>CTIP2.</t> ( B–C ) The percentage of CTIP2-positive cells was similar in different transplanted animals (example showing five different animals) and between human and chimpanzee transplanted cells (mean of 71% of co-localization). ( D ) Images of neurons transplanted in mouse cortex (arrowheads) showing absence of co-localization with upper cortical layer marker (SATB2).
    Ctip2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam ctip2 fitc 25b6 ab123449
    In vivo differentiation of human and chimpanzee neurons post-transplantation. ( A ) Images of pyramidal neurons (arrowheads) transplanted in mouse cortex showing co-localization with deep layer (layer V-VI) cortical marker <t>CTIP2.</t> ( B–C ) The percentage of CTIP2-positive cells was similar in different transplanted animals (example showing five different animals) and between human and chimpanzee transplanted cells (mean of 71% of co-localization). ( D ) Images of neurons transplanted in mouse cortex (arrowheads) showing absence of co-localization with upper cortical layer marker (SATB2).
    Ctip2 Fitc 25b6 Ab123449, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bethyl anti ctip2
    HMGA1 interacts with <t>CTIP2</t> in an RNA-independent fashion, while the association with inactive P-TEFb is RNase-sensitive. ( A ) Immunopurification of HMGA1-FLAG from transiently transfected HEK293 cells in the presence or the absence of RNase A. Input and precipitation steps were monitored for endogenous CTIP2, Cdk9, CycT1, HEXIM1 and the 7SK-associated LARP7 in western blot analyses. Mock-transfected cells were used as a control. ( B ) Immunopurification of endogenous HMGA1 from microglial cells using a specific anti-HMGA1 antibody. Input and precipitation steps were monitored for endogenous HMGA1 and CTIP2. An immunopurification with boiled anti-HMGA1 IgG was used as a control.
    Anti Ctip2, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bethyl ctip2
    LSD1 cooperates with <t>CTIP2</t> to repress HIV-1 gene transcription and viral replication. ( A ) Microglial cells were transfected with pNL-4.3 and the indicated plasmids (columns 2 to 4) or the pshRNA-control vector (column 1). Culture supernatants were analysed for p24 contents 48 h post-transfection. ( B and C ) Microglial cells were transfected with the episomal LTR-LUC and the indicated plasmids or the pshRNA-control vector. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with the episomal LTR-LUC and the control vectors (columns 1). DNA quantities were normalized with the pshRNA-control vector. ( D ) The knock-down efficiency of sh-RNA constructs was controlled by western blot analysis. The control columns 1 of the panels correspond to extracts from cells transfected with the pshRNA-control vector.
    Ctip2, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology ctip2
    COUP-TF-interacting protein 2 <t>(CTIP2)</t> impairs Vpr-mediated stimulation of p21 gene transcription and the resulting cell cycle arrest. ( a ) 293T cells inducible for HIV-1 Vpr expression, were transfected with the Flag-CTIP2 expression vector or the control
    Ctip2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Differentiation of glutamatergic and GABAergic neurons from dorsal and ventral progenitors. ( A , B ) Comparison of the expression of TBR1, CTIP2 and ISLET1 between the neurons that were developed from dorsal (control group) and ventral (DKK1 plus SHH group)

    Journal:

    Article Title: Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells

    doi: 10.1242/dev.036624

    Figure Lengend Snippet: Differentiation of glutamatergic and GABAergic neurons from dorsal and ventral progenitors. ( A , B ) Comparison of the expression of TBR1, CTIP2 and ISLET1 between the neurons that were developed from dorsal (control group) and ventral (DKK1 plus SHH group)

    Article Snippet: The primary antibodies used in this study included GAD65/67 (1:5000, rabbit IgG, Chemicon), GABA (1:5000, rabbit IgG, Chemicon), DARPP32 (1:500, rabbit IgG, Chemicon), vesicular glutamate transporter 1 (1:1000, rabbit IgG, Synaptic Systems), TBR1 (1:2000, rabbit IgG, Chemicon), NKX2-1 (1:200, mouse IgG, Chemicon), βIII-tubulin (1:5000, rabbit IgG, Covance), CTIP2 (1:2000, rat IgG, Chemicon), mono-βIII-tubulin (1:1000, mouse IgG, Sigma), PAX6 [1:5000, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA] and HOXB4 (1:50, DSHB).

    Techniques: Expressing

    Nrg1 expression in cingulate cortex–BLA circuit: heterozygous deletion of type III Nrg1 alters properties of miniature EPSCs in BLA pyramidal neurons. A , Representative confocal image of Nrg-1 (red) and CTIP-2 expression in the cingulate cortex.

    Journal: The Journal of Neuroscience

    Article Title: Type III Neuregulin 1 Is Required for Multiple Forms of Excitatory Synaptic Plasticity of Mouse Cortico-Amygdala Circuits

    doi: 10.1523/JNEUROSCI.2888-12.2013

    Figure Lengend Snippet: Nrg1 expression in cingulate cortex–BLA circuit: heterozygous deletion of type III Nrg1 alters properties of miniature EPSCs in BLA pyramidal neurons. A , Representative confocal image of Nrg-1 (red) and CTIP-2 expression in the cingulate cortex.

    Article Snippet: Immunostaining with specific antibodies against type III Nrg1 ( , , ; ) and CTIP-2, a transcription factor expressed by deep layer subcortical projection neurons ( ; ; ; ) revealed type III Nrg1 expression in cortical pyramidal neurons ( A ) and demonstrated a pronounced reduction in type III Nrg1 expression in these same type of cortical neurons from type III Nrg1 heterozygous animals ( B ).

    Techniques: Expressing

    Distribution of cortical neurons in the Smad4 conditional mutant mice. A) Brain sections of postnatal day 1 (P1) hGFAP-Cre;Smad4flx/- mutants and their control littermates were stained for Cux1, an upper layer neuronal marker, and Ctip2. Representative images obtained from the level of the corpus callosum (anterior) and the hippocampal commissure (posterior) are presented. The drawing in the upper panel shows the loss of the boundary between the Cux1 (red) and the Ctip2 (green) layers in mutants. A’) The numbers of Ctip2+ and Cux1+ neurons were plotted ( n = 6). A”) The distances between the pia and the Ctip2+ and Cux1+ neurons are shown ( n = 6). B) E17.5 Nex-Cre;Smad4flx/flx mutants were stained for Ctip2 and Map2. B’) The distances between the pia and the Ctip2+ neurons were plotted ( n = 6). The red and yellow dotted lines represent the pia and the Ctip2+ neurons closest to the pia, respectively. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Distribution of cortical neurons in the Smad4 conditional mutant mice. A) Brain sections of postnatal day 1 (P1) hGFAP-Cre;Smad4flx/- mutants and their control littermates were stained for Cux1, an upper layer neuronal marker, and Ctip2. Representative images obtained from the level of the corpus callosum (anterior) and the hippocampal commissure (posterior) are presented. The drawing in the upper panel shows the loss of the boundary between the Cux1 (red) and the Ctip2 (green) layers in mutants. A’) The numbers of Ctip2+ and Cux1+ neurons were plotted ( n = 6). A”) The distances between the pia and the Ctip2+ and Cux1+ neurons are shown ( n = 6). B) E17.5 Nex-Cre;Smad4flx/flx mutants were stained for Ctip2 and Map2. B’) The distances between the pia and the Ctip2+ neurons were plotted ( n = 6). The red and yellow dotted lines represent the pia and the Ctip2+ neurons closest to the pia, respectively. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Mutagenesis, Mouse Assay, Staining, Marker

    Cytoskeletal changes in cortical neurons induced by Bmp signaling through regulation of cofilin-1 activity. A) E15.5 cortices were used to establish cortical neuronal culture for 5 days in vitro , when more than 50% of the neurons were positive for Ctip2+ (data not shown). Cells were treated with Bmp7 (20 ng/ml) or Noggin (40 ng/ml) for 2 hr on day 6 or 24 hr on day 5 (A) and collected at the same time on day 6 ( n = 3). B) High-magnification images of representative cells are presented. The numbers of cells having motile or collapsed neurites (A”) were plotted for the 2 hr and 24 hr treatments (A’, B’). We defined a neuron as having collapsed neurites when it had more than two collapsed neurites, and we counted more than 100 neurons with fewer than 3 neurites. C) Cortical neurons (Tuj1+, red) cultured for 5 days in vitro were stained for phospho-Limk1/2 (pLimk1/2, green) or phospho-cofilin-1 (pcofilin-1, green) after treatment with Bmp7 (20 ng/ml) ( n = 3). Tuj1 signal is not presented separately. D) P1 hGFAP-Cre;Smad4flx/- mutants and their control littermates were stained for cofilin-1, Ctip2 (top panels) and pcofilin-1 and Ctip2 (middle panels). High-magnification images from the boxed areas in the middle panels are presented in the bottom panels ( n = 3). Student’s t -test was conducted to determine the statistical significance of the difference between the groups ( n = 3). Scale bars = 10 μm (A’, B), 50 μm (A, C), 100 μm (D).

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Cytoskeletal changes in cortical neurons induced by Bmp signaling through regulation of cofilin-1 activity. A) E15.5 cortices were used to establish cortical neuronal culture for 5 days in vitro , when more than 50% of the neurons were positive for Ctip2+ (data not shown). Cells were treated with Bmp7 (20 ng/ml) or Noggin (40 ng/ml) for 2 hr on day 6 or 24 hr on day 5 (A) and collected at the same time on day 6 ( n = 3). B) High-magnification images of representative cells are presented. The numbers of cells having motile or collapsed neurites (A”) were plotted for the 2 hr and 24 hr treatments (A’, B’). We defined a neuron as having collapsed neurites when it had more than two collapsed neurites, and we counted more than 100 neurons with fewer than 3 neurites. C) Cortical neurons (Tuj1+, red) cultured for 5 days in vitro were stained for phospho-Limk1/2 (pLimk1/2, green) or phospho-cofilin-1 (pcofilin-1, green) after treatment with Bmp7 (20 ng/ml) ( n = 3). Tuj1 signal is not presented separately. D) P1 hGFAP-Cre;Smad4flx/- mutants and their control littermates were stained for cofilin-1, Ctip2 (top panels) and pcofilin-1 and Ctip2 (middle panels). High-magnification images from the boxed areas in the middle panels are presented in the bottom panels ( n = 3). Student’s t -test was conducted to determine the statistical significance of the difference between the groups ( n = 3). Scale bars = 10 μm (A’, B), 50 μm (A, C), 100 μm (D).

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Activity Assay, In Vitro, Cell Culture, Staining

    Distribution of cortical neurons in the meninges-specific Bmp mutants. A) E15.5 embryonic brains of Wnt1-Cre;Bmp7flx/flx mutants and their control littermates were stained for Ctip2 or Satb2. A’) The distance of the pia and the Ctip2+ neurons were plotted ( n = 3). B) E17.5 embryonic brains of Wnt1-Cre;Bmp7flx/flx mutants and their control littermates were stained for Ctip2 or Satb2 ( n = 3). C) E15.5 embryonic brains of Pdgfrβ-Cre;Bmp4flx/flx mutants and their control littermates were stained for Satb2 or Ctip2. C’) The distances between the pia and the Ctip2+ neurons were plotted ( n = 3). The red and yellow dotted lines represent the pia and the Ctip2+ neurons closest to the pia, respectively. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Distribution of cortical neurons in the meninges-specific Bmp mutants. A) E15.5 embryonic brains of Wnt1-Cre;Bmp7flx/flx mutants and their control littermates were stained for Ctip2 or Satb2. A’) The distance of the pia and the Ctip2+ neurons were plotted ( n = 3). B) E17.5 embryonic brains of Wnt1-Cre;Bmp7flx/flx mutants and their control littermates were stained for Ctip2 or Satb2 ( n = 3). C) E15.5 embryonic brains of Pdgfrβ-Cre;Bmp4flx/flx mutants and their control littermates were stained for Satb2 or Ctip2. C’) The distances between the pia and the Ctip2+ neurons were plotted ( n = 3). The red and yellow dotted lines represent the pia and the Ctip2+ neurons closest to the pia, respectively. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Staining, Mutagenesis

    Distribution of cortical neurons in the Pdgfrβ-Cre;Foxc1flx/flx meningeal mutant. Embryonic brains were stained for Satb2 or Ctip2 at E15.5 (A) and E17.5 (B). The numbers of Ctip2+ and Satb2+ neurons were plotted ( n = 4, A’, B’). The distances between the pia and the Ctip2+ or Satb2+ neurons closest to the pia were plotted ( n = 4, A”, B”) (refer to the drawing in C). Error bars represent the SEM. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. The red dotted lines and the yellow dotted lines represent the pia and the sixth-closest Ctip2+ neurons to the pia, respectively. Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Distribution of cortical neurons in the Pdgfrβ-Cre;Foxc1flx/flx meningeal mutant. Embryonic brains were stained for Satb2 or Ctip2 at E15.5 (A) and E17.5 (B). The numbers of Ctip2+ and Satb2+ neurons were plotted ( n = 4, A’, B’). The distances between the pia and the Ctip2+ or Satb2+ neurons closest to the pia were plotted ( n = 4, A”, B”) (refer to the drawing in C). Error bars represent the SEM. Student’s t -test was conducted to determine the statistical significance of the difference between the control and mutant embryos. The red dotted lines and the yellow dotted lines represent the pia and the sixth-closest Ctip2+ neurons to the pia, respectively. Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Mutagenesis, Staining

    Effect of Bmp signaling on the cortical neuronal distribution. In utero electroporation with Bmp7, Noggin, Tgfβ1, and Follistatin (Fst) and IRES-driven eGFP constructs was conducted at E14.5 in CD1 mice. Three days after the electroporation, eGFP-expressing neurons were co-stained for Ctip2 (A) or Satb2 (B). A’, B’) Intensities of Ctip2+ and Satb2+ neurons in the electroporated field were plotted using the plot profile function in ImageJ software, and results from one representative experiment are shown ( n = 3). For a control image of Satb2, the contralateral side of the electroporated brain (A) is used. Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Effect of Bmp signaling on the cortical neuronal distribution. In utero electroporation with Bmp7, Noggin, Tgfβ1, and Follistatin (Fst) and IRES-driven eGFP constructs was conducted at E14.5 in CD1 mice. Three days after the electroporation, eGFP-expressing neurons were co-stained for Ctip2 (A) or Satb2 (B). A’, B’) Intensities of Ctip2+ and Satb2+ neurons in the electroporated field were plotted using the plot profile function in ImageJ software, and results from one representative experiment are shown ( n = 3). For a control image of Satb2, the contralateral side of the electroporated brain (A) is used. Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: In Utero, Electroporation, Construct, Mouse Assay, Expressing, Staining, Software

    Rescue of neuronal migration defects of Smad4 mutant neurons by cofilin-1-S3D expression. E13.5 Smad4flx/flx embryos were electroporated with 1) T1α-GFP, 2) T1α-Cre;Z/EG, or 3) T1α-Cre;cofilin-1-S3D;Z/EG, a Cre reporter expressing GFP after recombination, and analyzed at E18.5 (A, A’) and P2 (B, B’) ( n = 3). Electroporated brains were stained for GFP and Ctip2, and GFP+ neurons in the upper layer above the Ctip2+ layer were counted. Student’s t -test was conducted to determine the statistical significance of the difference between the groups. Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Rescue of neuronal migration defects of Smad4 mutant neurons by cofilin-1-S3D expression. E13.5 Smad4flx/flx embryos were electroporated with 1) T1α-GFP, 2) T1α-Cre;Z/EG, or 3) T1α-Cre;cofilin-1-S3D;Z/EG, a Cre reporter expressing GFP after recombination, and analyzed at E18.5 (A, A’) and P2 (B, B’) ( n = 3). Electroporated brains were stained for GFP and Ctip2, and GFP+ neurons in the upper layer above the Ctip2+ layer were counted. Student’s t -test was conducted to determine the statistical significance of the difference between the groups. Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Migration, Mutagenesis, Expressing, Staining

    Activation of Bmp signaling in the postmitotic cortical neurons. A) Double staining of pSmad1/5/8 and Satb2 or Ctip2 during cortical neurogenesis. The same section was used to triple label pSmad1/5/8, Satb2 and Ctip2, and separate images are presented to show Satb2- and Ctip2-expressing neurons (red) in the presence of the same pSmad1/5/8 signals (green) in a tissue. A’) Double-stained cells were plotted. Error bars represent the SEM. Student’s t -test was conducted to determine the statistical significance of the difference between pSmad1/5/8+;Ctip2+ and pSmad1/5/8+; Satb2+ n = 3). Scale bars = 100 μm.

    Journal: Brain Plasticity

    Article Title: Meningeal Bmps Regulate Cortical Layer Formation

    doi: 10.3233/BPL-170048

    Figure Lengend Snippet: Activation of Bmp signaling in the postmitotic cortical neurons. A) Double staining of pSmad1/5/8 and Satb2 or Ctip2 during cortical neurogenesis. The same section was used to triple label pSmad1/5/8, Satb2 and Ctip2, and separate images are presented to show Satb2- and Ctip2-expressing neurons (red) in the presence of the same pSmad1/5/8 signals (green) in a tissue. A’) Double-stained cells were plotted. Error bars represent the SEM. Student’s t -test was conducted to determine the statistical significance of the difference between pSmad1/5/8+;Ctip2+ and pSmad1/5/8+; Satb2+ n = 3). Scale bars = 100 μm.

    Article Snippet: This result clearly shows that most of the Ctip2+ deep-layer neurons responded to Bmp signaling and that 60% of Satb2+ neurons were pSmad1/5/8+ during embryonic cortical development, implying that Bmp signaling may function during the formation of cortical layers ( , p = 0.0049 (E14.5), p < 0.0001 (E16.5) comparison of pSmad1/5/8+, Ctip2+ vs. pSmad1/5/8+, Satb2+ cells (n = 3)).

    Techniques: Activation Assay, Double Staining, Expressing, Staining

    The striatal development gene expression signature is applied to evaluate the efficiency of human pluripotent stem cell (hPSC) differentiation toward a striatal fate. ( a ) Differentiation phases from pluripotent stem cells (PSCs) to medium spiny neurons (MSNs). Stem cell differentiation protocols translate in vivo development into an in vitro system. PSC-derived neurons are obtained via a three-step protocol. The first step is neuroectodermal induction (neuralization) that generates neural progenitors. The second step confers rostro-caudal and dorso-ventral patterning occurs and the final step involves the neurogenesis and differentiation of neuronal subtypes. This is a representation of how this three-step process mimics subpallial and then striatal development to generate MSNs. ( b ) Unbiased hierarchical clustering of differentiated hPSCs with human whole ganglionic eminence (WGE) and adult striatum. We isolated RNA from differentiating human PSCs at six time points (shown in panel a ) to analyze the progress of the differentiation protocol by high-throughput quantitative real-time PCR. We compared the gene expression profile of differentiated hPSCs (H9 and hiPSC) to human WGE and adult striatal tissue in order to establish the spatio-temporal gene expression pattern of iPSC-derived neural progenitors and neurons. Striatal signature genes with PPP1R1B and BCL11B are indicated in the heat map. ( c ) Representative immunofluorescence images of hESCs (H9) and hiPSCs (CS83iCTR33n1) during differentiation from pluripotency to striatal neurons. H9 cells were differentiated until 16 DIV, while hiPSCs were differentiated to 28 DIV. At 4 DIV, the majority of cells had a neuroectodermal identity as shown by the PAX6 staining. At 8DIV, a neuroepithelial morphology was observed with the cells organizing in rosettes, expressing nuclear marker PLZF and displaying polarized ZO1 distribution. At 16 DIV, subpallial progenitors expressed DLX1 and give rise to β-III tubulin positive neurons at 23 DIV. At 28 DIV, the presence of DARPP-32/CTIP2 (MSN markers at the protein level) double-positive neurons is observed. CGE, caudal ganglionic eminence; DIV, days in vitro ; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

    doi: 10.1038/mtm.2015.30

    Figure Lengend Snippet: The striatal development gene expression signature is applied to evaluate the efficiency of human pluripotent stem cell (hPSC) differentiation toward a striatal fate. ( a ) Differentiation phases from pluripotent stem cells (PSCs) to medium spiny neurons (MSNs). Stem cell differentiation protocols translate in vivo development into an in vitro system. PSC-derived neurons are obtained via a three-step protocol. The first step is neuroectodermal induction (neuralization) that generates neural progenitors. The second step confers rostro-caudal and dorso-ventral patterning occurs and the final step involves the neurogenesis and differentiation of neuronal subtypes. This is a representation of how this three-step process mimics subpallial and then striatal development to generate MSNs. ( b ) Unbiased hierarchical clustering of differentiated hPSCs with human whole ganglionic eminence (WGE) and adult striatum. We isolated RNA from differentiating human PSCs at six time points (shown in panel a ) to analyze the progress of the differentiation protocol by high-throughput quantitative real-time PCR. We compared the gene expression profile of differentiated hPSCs (H9 and hiPSC) to human WGE and adult striatal tissue in order to establish the spatio-temporal gene expression pattern of iPSC-derived neural progenitors and neurons. Striatal signature genes with PPP1R1B and BCL11B are indicated in the heat map. ( c ) Representative immunofluorescence images of hESCs (H9) and hiPSCs (CS83iCTR33n1) during differentiation from pluripotency to striatal neurons. H9 cells were differentiated until 16 DIV, while hiPSCs were differentiated to 28 DIV. At 4 DIV, the majority of cells had a neuroectodermal identity as shown by the PAX6 staining. At 8DIV, a neuroepithelial morphology was observed with the cells organizing in rosettes, expressing nuclear marker PLZF and displaying polarized ZO1 distribution. At 16 DIV, subpallial progenitors expressed DLX1 and give rise to β-III tubulin positive neurons at 23 DIV. At 28 DIV, the presence of DARPP-32/CTIP2 (MSN markers at the protein level) double-positive neurons is observed. CGE, caudal ganglionic eminence; DIV, days in vitro ; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence.

    Article Snippet: Cells were incubated overnight at 4 °C with 7% normal goat serum (S-1000, VectorLabs, Peterborough, UK) in PBS containing 0.3% Triton ×100, 1% Bovine serum albumin (BSA), 0.3% sodium azide and primary antibodies: mouse anti-OCT4 (1:100, sc-5279, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-PAX6 (1:200; Hybridoma Bank, Iowa City, Iowa), mouse anti-PLZF (1:100, OP128, Calbiochem, Madrid, Spain), rabbit anti-ZO1 (1:100, 40–2200, Invitrogen, Barcelona, Spain), rabbit anti-DLX (1:200, PA5967, CHDI, Princeton, NJ), anti-rabbit DARPP-32 (1:100, sc-11365, Santa Cruz Biotechnology, Heidelberg, Germany), anti-rat CTIP2 (1:300, Abcam ab18465), anti-rabbit β-III tubulin (1:1000, T2200, Sigma-Aldrich, Madrid, Spain).

    Techniques: Expressing, Cell Differentiation, In Vivo, In Vitro, Derivative Assay, Isolation, High Throughput Screening Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Marker

    Quantification of c-Fos+ cells in the frontal cortex of chronic saline and PCP-treated mice. (A) Schematic illustration of brain sampling schedule for brain-wide c-Fos mapping after working memory task. The timing of sampling for c-Fos immunohistochemistry is indicated with the red dashed lines. (B) Representative c-Fos staining (red) in the PL (B, top). Representative double staining for a layer 5 (and 6) marker, Ctip2 (magenta), and a layer 6 marker, Foxp2 (green), in sections adjacent to those used for c-Fos immunostaining in the PL (B, bottom). Scale bar, 100 μm. (C) Fluorescence microscopy images of c-Fos staining (red) in the frontal cortex of chronic saline- or PCP-treated mice. Scale bars, 100 μm. (D) Quantification of the total number of c-Fos+ cells in the ACC, PL, and IL of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5). (E–G) Quantitative laminar-specific c-Fos mapping in the ACC (E), PL (F) and IL (G). * p

    Journal: PLoS ONE

    Article Title: Abnormal neural activation patterns underlying working memory impairment in chronic phencyclidine-treated mice

    doi: 10.1371/journal.pone.0189287

    Figure Lengend Snippet: Quantification of c-Fos+ cells in the frontal cortex of chronic saline and PCP-treated mice. (A) Schematic illustration of brain sampling schedule for brain-wide c-Fos mapping after working memory task. The timing of sampling for c-Fos immunohistochemistry is indicated with the red dashed lines. (B) Representative c-Fos staining (red) in the PL (B, top). Representative double staining for a layer 5 (and 6) marker, Ctip2 (magenta), and a layer 6 marker, Foxp2 (green), in sections adjacent to those used for c-Fos immunostaining in the PL (B, bottom). Scale bar, 100 μm. (C) Fluorescence microscopy images of c-Fos staining (red) in the frontal cortex of chronic saline- or PCP-treated mice. Scale bars, 100 μm. (D) Quantification of the total number of c-Fos+ cells in the ACC, PL, and IL of chronic saline- (0 min: n = 4, 90 min: n = 5) and PCP-treated mice (0 min: n = 4, 90 min: n = 5). (E–G) Quantitative laminar-specific c-Fos mapping in the ACC (E), PL (F) and IL (G). * p

    Article Snippet: To identify laminar cytoarchitecture in the cerebral cortex, sections adjacent to those used for c-Fos immunostaining were used for Ctip2 staining (1:500, ab18465, abcam) as a marker for cortical layer 5 (and 6) and Foxp2 staining (1:2000, ab16046, abcam) as a marker for cortical layer 6.

    Techniques: Mouse Assay, Sampling, Immunohistochemistry, Staining, Double Staining, Marker, Immunostaining, Fluorescence, Microscopy

    Activity-regulated gene expression is strongly suppressed in ERK-deleted D2R-MSNs. A , Representative image of cataleptic response to haloperidol using horizontal bar test. B , Quantification of cataleptic response (freezing) to haloperidol (1 mg/kg) or vehicle in littermate control and ERK:D2 mutant mice. Control mice exhibit a robust cataleptic response to haloperidol compared with vehicle-treated controls (* p = 0.001, n = 7 mice/genotype/condition). The cataleptic response is effectively abolished in the mutant mice (* p = 0.001, haloperidol-treated ERK:D2 vs haloperidol treated control; n = 7 mice/condition). Vehicle-treated mice show no cataleptic response ( n = 7 mice per genotype per treatment). C , D , c-FOS (green) expression in control and ERK:D2 mutant striatum 1 h after haloperidol administration. CTIP2 (blue) identifies all MSNs; D1 tdTomato (red) identifies the D1R-MSN subpopulation. All D2R-MSNs are CTIP2(+);tdTomato(−). Insets are magnified images showing all three labels (top) and c-FOS only (bottom) demonstrating that c-FOS is strongly upregulated in D2R-MSNs (yellow arrows) in control ( C ) but not ERK:D2 animals ( D ). E , Quantification of MSN-specific c-FOS expression after haloperidol (1 mg/kg) or vehicle administration (* p

    Journal: The Journal of Neuroscience

    Article Title: ERK/MAPK Signaling Is Required for Pathway-Specific Striatal Motor Functions

    doi: 10.1523/JNEUROSCI.0473-17.2017

    Figure Lengend Snippet: Activity-regulated gene expression is strongly suppressed in ERK-deleted D2R-MSNs. A , Representative image of cataleptic response to haloperidol using horizontal bar test. B , Quantification of cataleptic response (freezing) to haloperidol (1 mg/kg) or vehicle in littermate control and ERK:D2 mutant mice. Control mice exhibit a robust cataleptic response to haloperidol compared with vehicle-treated controls (* p = 0.001, n = 7 mice/genotype/condition). The cataleptic response is effectively abolished in the mutant mice (* p = 0.001, haloperidol-treated ERK:D2 vs haloperidol treated control; n = 7 mice/condition). Vehicle-treated mice show no cataleptic response ( n = 7 mice per genotype per treatment). C , D , c-FOS (green) expression in control and ERK:D2 mutant striatum 1 h after haloperidol administration. CTIP2 (blue) identifies all MSNs; D1 tdTomato (red) identifies the D1R-MSN subpopulation. All D2R-MSNs are CTIP2(+);tdTomato(−). Insets are magnified images showing all three labels (top) and c-FOS only (bottom) demonstrating that c-FOS is strongly upregulated in D2R-MSNs (yellow arrows) in control ( C ) but not ERK:D2 animals ( D ). E , Quantification of MSN-specific c-FOS expression after haloperidol (1 mg/kg) or vehicle administration (* p

    Article Snippet: Primary antibodies used for immunohistochemistry were as follows: rabbit Erk2 and rat Ctip2 (1:500, Abcam), rabbit c-FOS (1:500; Cell Signaling Technology), chicken GFP (1:1000; Aves Laboratories), rabbit RFP and mouse RFP (1:250; Rockland), and rabbit ARC (1:1000; Synaptic Systems).

    Techniques: Activity Assay, Expressing, Mutagenesis, Mouse Assay

    Delayed loss of ERK activity in ERK:A2a mice. A , Representative image of P21 ERK:A2a striatum. CTIP2 (blue) labels all MSNs, whereas D2R-MSNs are identified as negative for D1 tdTomato expression (red). A subpopulation of D2R-MSNs in ERK:A2a mutants maintain ERK2 expression (green, white arrows). ERK2-deficient D2R-MSNs are indicated with a white asterisk. Scale bar, 20 μm. B , Quantitative analysis of ERK2 expression in P21 and P28 ERK:A2a striatum. Approximately half of all D2R-MSNs maintain ERK2 expression at P21. By 28, ∼15% of D2R-MSNs continue to express ERK2. ( n = 3 animals/genotype; 500–600cells/animal). C , Colocalization of D2 GFP (green) and A2a Cre :Ai9 (red) at P14. Scale bar, 50 μm. D , Quantification of D2 GFP and A2a Cre :Ai9 colocalization at P14, P21, and P28. All data are presented as a percentage of total D2R-MSNs counted. Note that the percentage of D2R-MSNs that express both GFP and Ai9 is low at P14. The percentage increases over time, but complete recombination in D2R-MSNs is not observed, even by P28 ( n = 3 animals/time point; 500–600 cells/animal). All data are presented as mean ± SEM.

    Journal: The Journal of Neuroscience

    Article Title: ERK/MAPK Signaling Is Required for Pathway-Specific Striatal Motor Functions

    doi: 10.1523/JNEUROSCI.0473-17.2017

    Figure Lengend Snippet: Delayed loss of ERK activity in ERK:A2a mice. A , Representative image of P21 ERK:A2a striatum. CTIP2 (blue) labels all MSNs, whereas D2R-MSNs are identified as negative for D1 tdTomato expression (red). A subpopulation of D2R-MSNs in ERK:A2a mutants maintain ERK2 expression (green, white arrows). ERK2-deficient D2R-MSNs are indicated with a white asterisk. Scale bar, 20 μm. B , Quantitative analysis of ERK2 expression in P21 and P28 ERK:A2a striatum. Approximately half of all D2R-MSNs maintain ERK2 expression at P21. By 28, ∼15% of D2R-MSNs continue to express ERK2. ( n = 3 animals/genotype; 500–600cells/animal). C , Colocalization of D2 GFP (green) and A2a Cre :Ai9 (red) at P14. Scale bar, 50 μm. D , Quantification of D2 GFP and A2a Cre :Ai9 colocalization at P14, P21, and P28. All data are presented as a percentage of total D2R-MSNs counted. Note that the percentage of D2R-MSNs that express both GFP and Ai9 is low at P14. The percentage increases over time, but complete recombination in D2R-MSNs is not observed, even by P28 ( n = 3 animals/time point; 500–600 cells/animal). All data are presented as mean ± SEM.

    Article Snippet: Primary antibodies used for immunohistochemistry were as follows: rabbit Erk2 and rat Ctip2 (1:500, Abcam), rabbit c-FOS (1:500; Cell Signaling Technology), chicken GFP (1:1000; Aves Laboratories), rabbit RFP and mouse RFP (1:250; Rockland), and rabbit ARC (1:1000; Synaptic Systems).

    Techniques: Activity Assay, Mouse Assay, Expressing

    Paxillin deficiency in post-mitotic neurons disrupts neuronal positioning. (A) Representative sections of littermate control ( Pxn F/F ) and mutant ( NEX-Cre:Pxn F/F ), with a conditional deletion of paxillin in post-mitotic immature neurons, were immunostained for Cux1 (green). More Cux1 + neurons were found in ectopic deep positions in the mutant cortex (arrow). (B) Box-and-whisker plot distribution of positions of Cux1 + neurons. The mutant showed a broader distribution compared with littermate controls. The mean position of Cux1 + neurons was significantly deeper in the mutant cortex ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ). (C) Representative images of Tle4 + (red) and Ctip2 + (green) neurons. (D) Distribution of Tbr1 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ), Tle4 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) and Ctip2 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) neurons. There was no difference in the mean positions of the Tbr1 + , Tle4 + and Ctip2 + neurons between the genotypes. *** P

    Journal: Development (Cambridge, England)

    Article Title: Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation

    doi: 10.1242/dev.147934

    Figure Lengend Snippet: Paxillin deficiency in post-mitotic neurons disrupts neuronal positioning. (A) Representative sections of littermate control ( Pxn F/F ) and mutant ( NEX-Cre:Pxn F/F ), with a conditional deletion of paxillin in post-mitotic immature neurons, were immunostained for Cux1 (green). More Cux1 + neurons were found in ectopic deep positions in the mutant cortex (arrow). (B) Box-and-whisker plot distribution of positions of Cux1 + neurons. The mutant showed a broader distribution compared with littermate controls. The mean position of Cux1 + neurons was significantly deeper in the mutant cortex ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ). (C) Representative images of Tle4 + (red) and Ctip2 + (green) neurons. (D) Distribution of Tbr1 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ), Tle4 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) and Ctip2 + ( n =3 in Pxn F/F and n =4 in NEX-Cre:Pxn F/F ) neurons. There was no difference in the mean positions of the Tbr1 + , Tle4 + and Ctip2 + neurons between the genotypes. *** P

    Article Snippet: Free-floating sections were immunostained ( ) using the following primary antibodies: anti-Cux1 (1:50, SantaCruz, sc-13024), anti-Tbr1 (1:500, EMD Millipore, AB2261), anti-Tle4 (1:1000; a kind gift from Dr Stefano Stifani, McGill University, Canada), anti-Ctip1 (1:500, Abcam, ab19489), anti-Ctip2 (1:500, Abcam, ab18465), anti-BrdU (1:10, DSHB) and anti-paxillin (1:200, rabbit monoclonal; clone Y113, Abcam).

    Techniques: Mutagenesis, Whisker Assay

    Combined Hic-5 deficiency with paxillin deficiency (dKO) produces an upper layer neuronal positioning defect similar to that observed with paxillin deficiency alone. (A-D) Neuronal position analysis of P0 cortex. (A) Representative images of P0 littermate control and dKO immunostained for Cux1 (green). More Cux1 + neurons were found in ectopic deep positions in the dKO cortex compared with littermate controls. (B) Box-and-whisker plot distribution of Cux1 + neurons showing a broad distribution of Cux1 + neurons in the dKO cortex. Mean position of Cux1 + neurons is significantly deeper in the dKO cortex ( n =5 per group). (C) Representative images of Tle4 + (red) and Ctip2 (green) neurons at P0. (D) Distribution of Tbr1 + ( n =5), Tle4 + ( n =4) and Ctip2 + ( n =3) neurons. No difference in the mean positions of the markers was found between genotypes. (E-H) Neuronal position analysis of P35 cortex. (E) Representative images of P35 littermate control and dKO immunostained for Cux1 (green). (F) Distribution of Cux1 + neurons. There was no difference in the mean position of Cux1 + neurons between genotypes ( n =6). (G) Representative image of Tle4 + (red) neurons at P35. (H) Distribution of Tbr1 + ( n =6) and Tle4 + ( n =4) neurons. There was no difference in the mean position of either Tbr1 + or Tle4 + neurons between genotypes. Data were analyzed using unpaired Student's t -test. * P

    Journal: Development (Cambridge, England)

    Article Title: Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation

    doi: 10.1242/dev.147934

    Figure Lengend Snippet: Combined Hic-5 deficiency with paxillin deficiency (dKO) produces an upper layer neuronal positioning defect similar to that observed with paxillin deficiency alone. (A-D) Neuronal position analysis of P0 cortex. (A) Representative images of P0 littermate control and dKO immunostained for Cux1 (green). More Cux1 + neurons were found in ectopic deep positions in the dKO cortex compared with littermate controls. (B) Box-and-whisker plot distribution of Cux1 + neurons showing a broad distribution of Cux1 + neurons in the dKO cortex. Mean position of Cux1 + neurons is significantly deeper in the dKO cortex ( n =5 per group). (C) Representative images of Tle4 + (red) and Ctip2 (green) neurons at P0. (D) Distribution of Tbr1 + ( n =5), Tle4 + ( n =4) and Ctip2 + ( n =3) neurons. No difference in the mean positions of the markers was found between genotypes. (E-H) Neuronal position analysis of P35 cortex. (E) Representative images of P35 littermate control and dKO immunostained for Cux1 (green). (F) Distribution of Cux1 + neurons. There was no difference in the mean position of Cux1 + neurons between genotypes ( n =6). (G) Representative image of Tle4 + (red) neurons at P35. (H) Distribution of Tbr1 + ( n =6) and Tle4 + ( n =4) neurons. There was no difference in the mean position of either Tbr1 + or Tle4 + neurons between genotypes. Data were analyzed using unpaired Student's t -test. * P

    Article Snippet: Free-floating sections were immunostained ( ) using the following primary antibodies: anti-Cux1 (1:50, SantaCruz, sc-13024), anti-Tbr1 (1:500, EMD Millipore, AB2261), anti-Tle4 (1:1000; a kind gift from Dr Stefano Stifani, McGill University, Canada), anti-Ctip1 (1:500, Abcam, ab19489), anti-Ctip2 (1:500, Abcam, ab18465), anti-BrdU (1:10, DSHB) and anti-paxillin (1:200, rabbit monoclonal; clone Y113, Abcam).

    Techniques: Hydrophobic Interaction Chromatography, Whisker Assay

    Overproduced CalR + cells were not cortical excitatory neurons. (A–B”) Immunostaining showed that CalR + cells were Satb2 - after Foxg1 was deleted at E12.5 and brains were analyzed at E18.5. (A”,B”) Show high magnification images of the boxed regions in (A’,B’) , respectively. (C–F”) Double staining of CalR with Ctip2 or Tbr1 showed that, compared with the controls (C–C”,E–E”) , a large proportion of CalR + cells located in the cortical plate co-expressed Ctip2 and Tbr1 (D–D”,F–F”) . (C”,D”,E”,F”) High magnification images of the boxed regions in (C’,D’,E’,F’) respectively. (G–H”) No co-localization of Foxp2 with CalR was detected in the mutant cortical plate after Foxg1 inactivation at E12.5. (G”,H”) Show high magnification images of the boxed regions in (G’,H’) , respectively. (I–J’) Foxg1 -ablated cells labeled with YFP rarely co-expressed Foxp2 in the mutants (J,J’) compared with the controls (I,I’) . (I’,J’) Show high magnification images of the boxed regions in (I”,J”) , respectively. (K) Statistical analysis of Satb2 + and Foxp2 + cells in the dorsal-lateral cortex (Ctip2, n = 3 mice per genotype, Control: 250.7 ± 4.343; CKO: 222.3 ± 13.05, ns, p = 0.1085; Tbr1, n = 3 mice per genotype, Control: 130.5 ± 7.522; CKO: 226.0 ± 9.504, ∗∗ p = 0.0014; Satb2, n = 3 mice per genotype, Control: 642.7 ± 23.69; CKO: 23.67 ± 4.419, ∗∗∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: FoxG1 Directly Represses Dentate Granule Cell Fate During Forebrain Development

    doi: 10.3389/fncel.2018.00452

    Figure Lengend Snippet: Overproduced CalR + cells were not cortical excitatory neurons. (A–B”) Immunostaining showed that CalR + cells were Satb2 - after Foxg1 was deleted at E12.5 and brains were analyzed at E18.5. (A”,B”) Show high magnification images of the boxed regions in (A’,B’) , respectively. (C–F”) Double staining of CalR with Ctip2 or Tbr1 showed that, compared with the controls (C–C”,E–E”) , a large proportion of CalR + cells located in the cortical plate co-expressed Ctip2 and Tbr1 (D–D”,F–F”) . (C”,D”,E”,F”) High magnification images of the boxed regions in (C’,D’,E’,F’) respectively. (G–H”) No co-localization of Foxp2 with CalR was detected in the mutant cortical plate after Foxg1 inactivation at E12.5. (G”,H”) Show high magnification images of the boxed regions in (G’,H’) , respectively. (I–J’) Foxg1 -ablated cells labeled with YFP rarely co-expressed Foxp2 in the mutants (J,J’) compared with the controls (I,I’) . (I’,J’) Show high magnification images of the boxed regions in (I”,J”) , respectively. (K) Statistical analysis of Satb2 + and Foxp2 + cells in the dorsal-lateral cortex (Ctip2, n = 3 mice per genotype, Control: 250.7 ± 4.343; CKO: 222.3 ± 13.05, ns, p = 0.1085; Tbr1, n = 3 mice per genotype, Control: 130.5 ± 7.522; CKO: 226.0 ± 9.504, ∗∗ p = 0.0014; Satb2, n = 3 mice per genotype, Control: 642.7 ± 23.69; CKO: 23.67 ± 4.419, ∗∗∗ p

    Article Snippet: The following antibodies and reagents were used in the present study: chicken anti-GFP (Abcam, AB13970, 1:1000); goat anti-CalR (Millipore, AB1550, 1:500); goat anti-Prox1 (R & D, AF2727, 1:250); mouse anti-Reelin (Millipore, MAB5364, 1:1000); mouse anti-Satb2 (Santa Cruz, SC81376, 1:500); rabbit anti-CalR (Millipore, AB5054, 1:1000); rabbit anti-FoxG1 (Abcam, AB18259, 1:250); rabbit anti-Foxp2 (Abcam, AB16046, 1:1000); rabbit anti-Lhx2 (Abcam, AB184337, 1:500); rabbit anti-Pax6 (Covance, 1:1000); rabbit anti-Tbr1 (Millipore, AB10554, 1:500); rat anti-Ctip2 (Abcam, AB18465, 1:1000); Alexa Fluor 488-conjugated goat anti-chicken (Invitrogen, A11039, 1:500); DyLight 488-conjugated donkey anti-rabbit (Thermo Fisher Scientific, SA5-10038, 1:500); DyLight 650-conjugated donkey anti-rabbit (Thermo Fisher Scientific, SA5-10041, 1:500); DyLight 488-conjugated donkey anti-goat (Thermo Fisher Scientific, SA5-10086, 1:500); Alexa Fluor 546-conjugated rabbit anti-goat (Invitrogen, A21085, 1:500); DyLight 488-conjugated donkey anti-mouse (Thermo Fisher Scientific, SA5-10066, 1:500); DyLight 550-conjugated donkey anti-mouse (Thermo Fisher Scientific, SA5-10067, 1:500); Alexa Fluor 488-conjugated goat anti-rat (Invitrogen, A11006, 1:500); Alexa Fluor 546-conjugated goat anti-rat (Invitrogen, A11081, 1:500); and DAPI (Sigma-Aldrich, D9564, 1:1000).

    Techniques: Immunostaining, Double Staining, Mutagenesis, Labeling, Mouse Assay

    Expression of Fezf2 in migratory, postmitotic CPN induces them to acquire molecular features of CFuPN a , Schematic representation of the experimental approach. Fezf2 or control vectors were electroporated at E14.5 and expression was restricted to CPN at the earliest stages of postmitotic development using the promoter of the Cdk5r gene. b , Quantification of the percentage of electroporated CPN expressing CFuPN-specific markers (ER81, CRYM, TLE4, CTIP2 and ZFPM2) and downregulating CPN-specific markers (CUX1). Results are expressed as the mean ± s.e.m. The paired, two-tailed t test was used for statistical analysis. * p

    Journal: Nature cell biology

    Article Title: Direct lineage reprogramming of postmitotic callosal neurons into corticofugal neurons in vivo

    doi: 10.1038/ncb2660

    Figure Lengend Snippet: Expression of Fezf2 in migratory, postmitotic CPN induces them to acquire molecular features of CFuPN a , Schematic representation of the experimental approach. Fezf2 or control vectors were electroporated at E14.5 and expression was restricted to CPN at the earliest stages of postmitotic development using the promoter of the Cdk5r gene. b , Quantification of the percentage of electroporated CPN expressing CFuPN-specific markers (ER81, CRYM, TLE4, CTIP2 and ZFPM2) and downregulating CPN-specific markers (CUX1). Results are expressed as the mean ± s.e.m. The paired, two-tailed t test was used for statistical analysis. * p

    Article Snippet: Primary antibodies and dilutions were as follows: rabbit anti-ZFPM2 antibody , 1:500 (Santa Cruz, sc-10755); rat anti-CTIP2 antibody, 1:1000 (Abcam, ab18465); rabbit anti-GFP antibody, 1:500 (Invitrogen, A-11122); rat anti-GFP antibody, 1:500 (Nacalai, 04404– 84); rabbit anti-ER81 antibody , 1:2500 (gift of Silvia Arber); rabbit anti-CRYM antibody , 1:100 (Abcam, ab54669); rabbit anti-TLE4 antibody , 1:100 (Santa Cruz, sc-9525); rabbit anti-cleaved Caspase-3, 1:400 (Cell Signaling Technology, 9661S).

    Techniques: Expressing, Two Tailed Test

    In vivo differentiation of human and chimpanzee neurons post-transplantation. ( A ) Images of pyramidal neurons (arrowheads) transplanted in mouse cortex showing co-localization with deep layer (layer V-VI) cortical marker CTIP2. ( B–C ) The percentage of CTIP2-positive cells was similar in different transplanted animals (example showing five different animals) and between human and chimpanzee transplanted cells (mean of 71% of co-localization). ( D ) Images of neurons transplanted in mouse cortex (arrowheads) showing absence of co-localization with upper cortical layer marker (SATB2).

    Journal: eLife

    Article Title: Species-specific maturation profiles of human, chimpanzee and bonobo neural cells

    doi: 10.7554/eLife.37527

    Figure Lengend Snippet: In vivo differentiation of human and chimpanzee neurons post-transplantation. ( A ) Images of pyramidal neurons (arrowheads) transplanted in mouse cortex showing co-localization with deep layer (layer V-VI) cortical marker CTIP2. ( B–C ) The percentage of CTIP2-positive cells was similar in different transplanted animals (example showing five different animals) and between human and chimpanzee transplanted cells (mean of 71% of co-localization). ( D ) Images of neurons transplanted in mouse cortex (arrowheads) showing absence of co-localization with upper cortical layer marker (SATB2).

    Article Snippet: CTIP2 antibody (1:200, Abcam) and SATB2 (1:200, Abcam) were used to characterize cortical layer identity of the transplanted cells ( ).

    Techniques: In Vivo, Transplantation Assay, Marker

    Immunocytochemistry of iPSC-derived neurons from humans and chimpanzees. ( A ) Representative images from neurons stained with pan neuronal marker (Map2) and cortical marker (CTIP2). Scale bar = 50 µm. ( B ) Quantification of the percentage of Map2-positive cells that express CITP2. No significant change was observed in the percentage of neurons expressing CTIP2 in vitro between human and chimpanzee cells.

    Journal: eLife

    Article Title: Species-specific maturation profiles of human, chimpanzee and bonobo neural cells

    doi: 10.7554/eLife.37527

    Figure Lengend Snippet: Immunocytochemistry of iPSC-derived neurons from humans and chimpanzees. ( A ) Representative images from neurons stained with pan neuronal marker (Map2) and cortical marker (CTIP2). Scale bar = 50 µm. ( B ) Quantification of the percentage of Map2-positive cells that express CITP2. No significant change was observed in the percentage of neurons expressing CTIP2 in vitro between human and chimpanzee cells.

    Article Snippet: CTIP2 antibody (1:200, Abcam) and SATB2 (1:200, Abcam) were used to characterize cortical layer identity of the transplanted cells ( ).

    Techniques: Immunocytochemistry, Derivative Assay, Staining, Marker, Expressing, In Vitro

    Phenotypic Similarity of Monosynaptic Inputs to Ectopic versus Homotopic VM-Patterned Grafts (A–F) CTIP2 + pyramidal neurons in motor cortex (A and B), MOR + medium spiny neurons in striatum (C and D), and PV + neurons in GPe (E, F) connected to both homotopic (intranigral) and ectopic (intrastriatal) grafts of VM-patterned neurons. (G) FOXP2 + GPe neurons were observed connecting only to grafts placed ectopically in the striatum. (H) Schematic representations of the origin of host synaptic inputs to grafts placed in the substantia nigra versus the striatum. Solid lines represent host brain structures with extensive synaptic input to transplanted neurons, while dashed lines represent structures with comparatively scarce inputs. Scale bars represent 20 μm. CTIP2, COUP-TF-interacting protein 2; FOXP2, forkhead box P2; GPe, external globus pallidus; MOR, mu-opioid receptor; PV, parvalbumin; SN, substantia nigra.

    Journal: Cell Reports

    Article Title: hESC-Derived Dopaminergic Transplants Integrate into Basal Ganglia Circuitry in a Preclinical Model of Parkinson’s Disease

    doi: 10.1016/j.celrep.2019.08.058

    Figure Lengend Snippet: Phenotypic Similarity of Monosynaptic Inputs to Ectopic versus Homotopic VM-Patterned Grafts (A–F) CTIP2 + pyramidal neurons in motor cortex (A and B), MOR + medium spiny neurons in striatum (C and D), and PV + neurons in GPe (E, F) connected to both homotopic (intranigral) and ectopic (intrastriatal) grafts of VM-patterned neurons. (G) FOXP2 + GPe neurons were observed connecting only to grafts placed ectopically in the striatum. (H) Schematic representations of the origin of host synaptic inputs to grafts placed in the substantia nigra versus the striatum. Solid lines represent host brain structures with extensive synaptic input to transplanted neurons, while dashed lines represent structures with comparatively scarce inputs. Scale bars represent 20 μm. CTIP2, COUP-TF-interacting protein 2; FOXP2, forkhead box P2; GPe, external globus pallidus; MOR, mu-opioid receptor; PV, parvalbumin; SN, substantia nigra.

    Article Snippet: Primary antibodies were used as follows: Goat anti-CTB (1:2500; List Biological Laboratories); rabbit anti-CTIP2 (1:1000; Abcam ab28448); rabbit anti-DARPP-32 (1:250 Cell Signaling 2306); goat anti-FOXA2 (1:500; Santa Cruz Biotechnology sc-6554); rabbit anti-FOXG1 (1:200; Abcam ab18259); mouse anti-FOXP2 (1:500; Millipore MABE415); chicken anti-GFP (1:1000; Abcam ab13970); mouse anti-hNCAM (1:1000; Santa Cruz Biotechnology sc-106); mouse anti-HuNu (1:200; Millipore MAB1281); goat anti-mCherry (1:1000; SICGEN AB0040-200); rabbit anti-mCherry (1:1000; Abcam ab167453); guinea pig anti-MOR (1:1000; Millipore ab5509); rabbit anti-NKX2.1 (aka TTF1; 1:250; Abcam ab133737) mouse anti-parvalbumin (1:2000; Sigma-Aldrich P3088); rabbit anti-TH (1:1000; Millipore AB152).

    Techniques:

    Expansion of cortical layer IV and depletion of cortical layers II–IV in the Atrx -null neocortex at P7. (A) TBR1 immunostaining in P7 control and Atrx -null cortical sections. Scale bar: 200 µm. (B) Quantification of TBR1 + layer VI thickness compared to total cortical thickness, the number, and proportion of TBR1 + layer VI neurons ( n = 5). (C) Western blot analysis of P10 control and Atrx -null forebrain tissue. α-Tubulin was used as a loading control. (D) CTIP2 and BRN2 immunostaining in P7 control and Atrx -null cortical sections. Scale bar: 200 µm. (E) Quantification of CTIP2 + layer V thickness compared to total cortical thickness, the number, and proportion of CTIP2 + layer V neurons ( n = 5). (F) Western blot analysis of P7 control and Atrx -null forebrain tissue. α-Tubulin was used as a loading control. (G) Quantification of BRN2 + cortical layer II–IV thickness compared to total cortical thickness, the number, and proportion of BRN2 + layer II–IV neurons ( n = 5). (H) Western blot analysis of P10 control and Atrx -null forebrain tissue. INCENP was used as a loading control. Data expressed as mean ± S.E.M. * P

    Journal: Biology Open

    Article Title: ATRX is required for maintenance of the neuroprogenitor cell pool in the embryonic mouse brain

    doi: 10.1242/bio.20148730

    Figure Lengend Snippet: Expansion of cortical layer IV and depletion of cortical layers II–IV in the Atrx -null neocortex at P7. (A) TBR1 immunostaining in P7 control and Atrx -null cortical sections. Scale bar: 200 µm. (B) Quantification of TBR1 + layer VI thickness compared to total cortical thickness, the number, and proportion of TBR1 + layer VI neurons ( n = 5). (C) Western blot analysis of P10 control and Atrx -null forebrain tissue. α-Tubulin was used as a loading control. (D) CTIP2 and BRN2 immunostaining in P7 control and Atrx -null cortical sections. Scale bar: 200 µm. (E) Quantification of CTIP2 + layer V thickness compared to total cortical thickness, the number, and proportion of CTIP2 + layer V neurons ( n = 5). (F) Western blot analysis of P7 control and Atrx -null forebrain tissue. α-Tubulin was used as a loading control. (G) Quantification of BRN2 + cortical layer II–IV thickness compared to total cortical thickness, the number, and proportion of BRN2 + layer II–IV neurons ( n = 5). (H) Western blot analysis of P10 control and Atrx -null forebrain tissue. INCENP was used as a loading control. Data expressed as mean ± S.E.M. * P

    Article Snippet: Antibodies used were goat anti-BRN2 (1:1000, Santa Cruz Biotechnology, Inc.), rabbit anti-TBR1 (1:1000, Abcam), and rabbit anti-CTIP2 (1:1000, Abcam) followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, GE Healthcare).

    Techniques: Immunostaining, Western Blot

    miR‐34/449 family is required for timely cortical neurogenesis Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Satb2‐antibody to label neurons of layers II–IV (green) and DAPI to label all cell nuclei (blue). Quantification of the number of Satb2‐positive cells per 100 μm ventricular zone surface ( n = 4 brains per genotype group, 2 independent litters). * P ‐value = 0.01146 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Ctip2 antibody to label V neurons and DAPI to label all cell nuclei (blue). Images were taken from same brain slices as shown in (A). Scale bars: 50 μm. Quantification of the number of Ctip2‐positive cells per 100 μm ventricular zone surface ( n = 4 brains per genotype group, 2 independent litters). * P ‐value = 0.03224 (Het vs. KO). Confocal images of coronal sections from E14 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr1 antibody to label layer IV neurons and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr1‐positive cells per 100 μm ventricular zone surface ( n = 3 and 4 brains per genotype group from 2 independent litters). * P ‐value = 0.01392 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr2 antibody to label intermediate progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2‐positive cells per 100 μm ventricular zone surface ( n = 7 brains for each genotype group, 5 independent litters). * P ‐value = 0.01647 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Pax6 antibody to label radial glia progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2‐positive cells per 100 μm ventricular zone surface ( n = 5 brains per genotype group, 3 independent litters). ** P ‐value = 0.002768 (Het vs. KO). 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti‐phospho‐vimentin antibody (red), anti‐γ‐tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. “s” indicates spindle axis, and “α” indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Quantification of spindle orientation in radial glial cells as in (K) for E14 brains of miR‐34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; *** P ‐value = 3.166e‐05 (Het vs. KO) ( n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters). Data information: Scale bars: 50 μm (A, C, E, G, I) and 5 μm (K). Bars indicate mean ± SEM. Data were normalized (norm.) to the ventricular zone surface analyzed (100 μm) and relativized to the heterozygous control average value. Statistical significance was tested by Welch's t ‐test. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Micro RNA‐34/449 controls mitotic spindle orientation during mammalian cortex development

    doi: 10.15252/embj.201694056

    Figure Lengend Snippet: miR‐34/449 family is required for timely cortical neurogenesis Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Satb2‐antibody to label neurons of layers II–IV (green) and DAPI to label all cell nuclei (blue). Quantification of the number of Satb2‐positive cells per 100 μm ventricular zone surface ( n = 4 brains per genotype group, 2 independent litters). * P ‐value = 0.01146 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Ctip2 antibody to label V neurons and DAPI to label all cell nuclei (blue). Images were taken from same brain slices as shown in (A). Scale bars: 50 μm. Quantification of the number of Ctip2‐positive cells per 100 μm ventricular zone surface ( n = 4 brains per genotype group, 2 independent litters). * P ‐value = 0.03224 (Het vs. KO). Confocal images of coronal sections from E14 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr1 antibody to label layer IV neurons and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr1‐positive cells per 100 μm ventricular zone surface ( n = 3 and 4 brains per genotype group from 2 independent litters). * P ‐value = 0.01392 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr2 antibody to label intermediate progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2‐positive cells per 100 μm ventricular zone surface ( n = 7 brains for each genotype group, 5 independent litters). * P ‐value = 0.01647 (Het vs. KO). Confocal images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Pax6 antibody to label radial glia progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2‐positive cells per 100 μm ventricular zone surface ( n = 5 brains per genotype group, 3 independent litters). ** P ‐value = 0.002768 (Het vs. KO). 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti‐phospho‐vimentin antibody (red), anti‐γ‐tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. “s” indicates spindle axis, and “α” indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Quantification of spindle orientation in radial glial cells as in (K) for E14 brains of miR‐34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; *** P ‐value = 3.166e‐05 (Het vs. KO) ( n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters). Data information: Scale bars: 50 μm (A, C, E, G, I) and 5 μm (K). Bars indicate mean ± SEM. Data were normalized (norm.) to the ventricular zone surface analyzed (100 μm) and relativized to the heterozygous control average value. Statistical significance was tested by Welch's t ‐test. Source data are available online for this figure.

    Article Snippet: The following primary antibodies were used: mouse anti‐γ‐tubulin (1:1,000, Sigma), rabbit anti‐Pax6 (1:250, Covance), rabbit anti‐caspase‐3 (1:250, Cell Signaling), chicken anti‐Ctip2 (1:250, Abcam), rabbit anti‐Tbr1 (1:250, Abcam), rabbit anti‐Tbr2 (1:250, Abcam), mouse anti‐Ki67 (1:100, Cell Signaling), and rabbit anti‐JAM‐A (aliquot 1168, Dr. Klaus Ebnet), as well as Alexa Fluor 647 phalloidin to stain actin (1:25, Life Technologies).

    Techniques: Mouse Assay, Staining, Confocal Microscopy, Plasmid Preparation

    miR‐34/449 family is required for timely cortical neurogenesis Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Satb2 antibody to label neurons of layers II–IV (green), anti‐Ctip2 antibody to label V neurons ( n = 4 brains per genotype group, 2 independent litters), and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E14 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr1 antibody to label layer IV neurons ( n = 3 brains per genotype group, 2 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr2 antibody to label intermediate progenitors ( n = 5 brains for each genotype group, 3 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Pax6 antibody to label radial glia progenitors ( n = 7 brains per genotype group, 5 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm.

    Journal: The EMBO Journal

    Article Title: Micro RNA‐34/449 controls mitotic spindle orientation during mammalian cortex development

    doi: 10.15252/embj.201694056

    Figure Lengend Snippet: miR‐34/449 family is required for timely cortical neurogenesis Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Satb2 antibody to label neurons of layers II–IV (green), anti‐Ctip2 antibody to label V neurons ( n = 4 brains per genotype group, 2 independent litters), and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E14 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr1 antibody to label layer IV neurons ( n = 3 brains per genotype group, 2 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Tbr2 antibody to label intermediate progenitors ( n = 5 brains for each genotype group, 3 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm. Confocal single channel images of coronal sections from E16 brains of miR‐34/449 KO mice and littermate controls (Het), stained with anti‐Pax6 antibody to label radial glia progenitors ( n = 7 brains per genotype group, 5 independent litters) and DAPI to label cell nuclei (blue). Scale bars: 50 μm.

    Article Snippet: The following primary antibodies were used: mouse anti‐γ‐tubulin (1:1,000, Sigma), rabbit anti‐Pax6 (1:250, Covance), rabbit anti‐caspase‐3 (1:250, Cell Signaling), chicken anti‐Ctip2 (1:250, Abcam), rabbit anti‐Tbr1 (1:250, Abcam), rabbit anti‐Tbr2 (1:250, Abcam), mouse anti‐Ki67 (1:100, Cell Signaling), and rabbit anti‐JAM‐A (aliquot 1168, Dr. Klaus Ebnet), as well as Alexa Fluor 647 phalloidin to stain actin (1:25, Life Technologies).

    Techniques: Mouse Assay, Staining

    HMGA1 interacts with CTIP2 in an RNA-independent fashion, while the association with inactive P-TEFb is RNase-sensitive. ( A ) Immunopurification of HMGA1-FLAG from transiently transfected HEK293 cells in the presence or the absence of RNase A. Input and precipitation steps were monitored for endogenous CTIP2, Cdk9, CycT1, HEXIM1 and the 7SK-associated LARP7 in western blot analyses. Mock-transfected cells were used as a control. ( B ) Immunopurification of endogenous HMGA1 from microglial cells using a specific anti-HMGA1 antibody. Input and precipitation steps were monitored for endogenous HMGA1 and CTIP2. An immunopurification with boiled anti-HMGA1 IgG was used as a control.

    Journal: Nucleic Acids Research

    Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

    doi: 10.1093/nar/gku168

    Figure Lengend Snippet: HMGA1 interacts with CTIP2 in an RNA-independent fashion, while the association with inactive P-TEFb is RNase-sensitive. ( A ) Immunopurification of HMGA1-FLAG from transiently transfected HEK293 cells in the presence or the absence of RNase A. Input and precipitation steps were monitored for endogenous CTIP2, Cdk9, CycT1, HEXIM1 and the 7SK-associated LARP7 in western blot analyses. Mock-transfected cells were used as a control. ( B ) Immunopurification of endogenous HMGA1 from microglial cells using a specific anti-HMGA1 antibody. Input and precipitation steps were monitored for endogenous HMGA1 and CTIP2. An immunopurification with boiled anti-HMGA1 IgG was used as a control.

    Article Snippet: The primary antibodies used for ChIP were anti-CTIP2 (bethyl), anti-Cdk9 (Santa Cruz), anti-Cyclin T1 (Santa Cruz) and anti-Hexim-1 (Abcam).

    Techniques: Immu-Puri, Transfection, Western Blot

    HMGA1 and CTIP2 cooperatively repress HIV-1 gene expression by a HMGA1-mediated recruitment of CTIP2-inactivated P-TEFb to the HIV promoter. ( A ) HIV-1 LTR-driven reporter assays in microglial cells on CTIP2 and/or HMGA1 knock-down. The magnitude of an additive effect of CTIP2 and HMGA1 knock-down is indicated as a dotted line. * , ** , *** in a Student’s t-test. ( B ) Transcription assays in HIV-latently infected CHME-5 cells on CTIP2 and/or HMGA1 knock-down. HIV gene expression was quantified by qRT-PCR and normalized to β-actin expression as a housekeeping gene. HIV gene expression on expression of a non-targeting control shRNA was arbitrarily set to 1. * , *** in a Student’s t-test. ( C ) As in (A), but in microglial cells expressing HIV-1 Tat-FLAG. ( D ) As in (A), but using the pNL4.3 delta ENV-luciferase proviral plasmid as reporter construct. ( E ) As in (D), but in microglial cells overexpressing CTIP2-FLAG and/or HMGA1-FLAG. Protein overexpression was verified in western blot experiments using anti-FLAG antibody. β-actin was used as a housekeeping gene. ( F ) ChIP analyses targeting the HIV-1 promoter from HEK293 cells on expression of HMGA1-FLAG (left panel) or knock-down of endogenous HMGA1 (right panel). ( G ) ChIP analyses targeting the HIV-1 promoter from HEK293 cells transfected with CTIP2-FLAG in the presence or absence of a HMGA1 knock-down (upper panel). The luciferase gene was used as a control (lower panel). ( H ) Model: The CTIP2-repressed 7SK/P-TEFb snRNP is recruited to cellular and viral promoters by the interaction of 7SK L2-bound HMGA1 with core promoter-bound basal transcription factors (TF, e.g. NF-κB or Sp1) or DNA.

    Journal: Nucleic Acids Research

    Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

    doi: 10.1093/nar/gku168

    Figure Lengend Snippet: HMGA1 and CTIP2 cooperatively repress HIV-1 gene expression by a HMGA1-mediated recruitment of CTIP2-inactivated P-TEFb to the HIV promoter. ( A ) HIV-1 LTR-driven reporter assays in microglial cells on CTIP2 and/or HMGA1 knock-down. The magnitude of an additive effect of CTIP2 and HMGA1 knock-down is indicated as a dotted line. * , ** , *** in a Student’s t-test. ( B ) Transcription assays in HIV-latently infected CHME-5 cells on CTIP2 and/or HMGA1 knock-down. HIV gene expression was quantified by qRT-PCR and normalized to β-actin expression as a housekeeping gene. HIV gene expression on expression of a non-targeting control shRNA was arbitrarily set to 1. * , *** in a Student’s t-test. ( C ) As in (A), but in microglial cells expressing HIV-1 Tat-FLAG. ( D ) As in (A), but using the pNL4.3 delta ENV-luciferase proviral plasmid as reporter construct. ( E ) As in (D), but in microglial cells overexpressing CTIP2-FLAG and/or HMGA1-FLAG. Protein overexpression was verified in western blot experiments using anti-FLAG antibody. β-actin was used as a housekeeping gene. ( F ) ChIP analyses targeting the HIV-1 promoter from HEK293 cells on expression of HMGA1-FLAG (left panel) or knock-down of endogenous HMGA1 (right panel). ( G ) ChIP analyses targeting the HIV-1 promoter from HEK293 cells transfected with CTIP2-FLAG in the presence or absence of a HMGA1 knock-down (upper panel). The luciferase gene was used as a control (lower panel). ( H ) Model: The CTIP2-repressed 7SK/P-TEFb snRNP is recruited to cellular and viral promoters by the interaction of 7SK L2-bound HMGA1 with core promoter-bound basal transcription factors (TF, e.g. NF-κB or Sp1) or DNA.

    Article Snippet: The primary antibodies used for ChIP were anti-CTIP2 (bethyl), anti-Cdk9 (Santa Cruz), anti-Cyclin T1 (Santa Cruz) and anti-Hexim-1 (Abcam).

    Techniques: Expressing, Infection, Quantitative RT-PCR, shRNA, Luciferase, Plasmid Preparation, Construct, Over Expression, Western Blot, Chromatin Immunoprecipitation, Transfection

    HMGA1 recruits CTIP2-inactivated P-TEFb to cellular target promoters. ( A ) Network of 7SK-dependent P-TEFb target genes affected by both knock-down of CTIP2 and HMGA1. Up-regulated genes are coloured in red, down-regulated genes are coloured in green. Functional connections are indicated as arrows. The inactive P-TEFb snRNP is highlighted in yellow (Cdk9, CycT1, HEXIM1, 7SK RNA). ( B ) The overexpression of CTIP2 (pCTIP2) was verified by western blot analyses and β-actin was used as a housekeeping gene. ( C ) Scatter plot comparing the expression changes of 7SK-dependent P-TEFb targets on CTIP2 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in blue. ( D ) The overexpression of HMGA1 (pHMGA1) was verified by western blot analyses, and β-actin was used as a housekeeping gene. ( E ) As in (C), but comparing HMGA1 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in red. ( F ) Representative visualization of the effects of dnCdk9 expression, 7SK overexpression, 7SK knock-down, CTIP2 knock-down, CTIP2 overexpression, HMGA1 knock-down and HMGA1 overexpression on the expression of the 7SK-dependent P-TEFb targets PARP12 and EPSTI1. ( G ) ChIP analyses targeting the EPSTI1 promoter from HEK293 cells transfected with CTIP2-FLAG in the presence or absence of a HMGA1 knock-down. ( H ) Same as in (G), but for the PARP12 promoter.

    Journal: Nucleic Acids Research

    Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

    doi: 10.1093/nar/gku168

    Figure Lengend Snippet: HMGA1 recruits CTIP2-inactivated P-TEFb to cellular target promoters. ( A ) Network of 7SK-dependent P-TEFb target genes affected by both knock-down of CTIP2 and HMGA1. Up-regulated genes are coloured in red, down-regulated genes are coloured in green. Functional connections are indicated as arrows. The inactive P-TEFb snRNP is highlighted in yellow (Cdk9, CycT1, HEXIM1, 7SK RNA). ( B ) The overexpression of CTIP2 (pCTIP2) was verified by western blot analyses and β-actin was used as a housekeeping gene. ( C ) Scatter plot comparing the expression changes of 7SK-dependent P-TEFb targets on CTIP2 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in blue. ( D ) The overexpression of HMGA1 (pHMGA1) was verified by western blot analyses, and β-actin was used as a housekeeping gene. ( E ) As in (C), but comparing HMGA1 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in red. ( F ) Representative visualization of the effects of dnCdk9 expression, 7SK overexpression, 7SK knock-down, CTIP2 knock-down, CTIP2 overexpression, HMGA1 knock-down and HMGA1 overexpression on the expression of the 7SK-dependent P-TEFb targets PARP12 and EPSTI1. ( G ) ChIP analyses targeting the EPSTI1 promoter from HEK293 cells transfected with CTIP2-FLAG in the presence or absence of a HMGA1 knock-down. ( H ) Same as in (G), but for the PARP12 promoter.

    Article Snippet: The primary antibodies used for ChIP were anti-CTIP2 (bethyl), anti-Cdk9 (Santa Cruz), anti-Cyclin T1 (Santa Cruz) and anti-Hexim-1 (Abcam).

    Techniques: Functional Assay, Over Expression, Western Blot, Expressing, Chromatin Immunoprecipitation, Transfection

    HMGA1 and CTIP2 repress the expression of 7SK-dependent P-TEFb target genes. ( A ) shRNA-mediated knock-down of CTIP2 in microglial cells. CTIP2 levels were monitored in western blot analyses and β-actin was used as a control (upper panel). Cell viability on shRNA-mediated knock-down was measured by MTT test (lower panel). n.s., not statistically significant in a students t -test. ( B ) shRNA-mediated knock-down of HMGA1 in microglial cells. HMGA1 amounts were quantified by western blot analyses, and β-actin was used as a housekeeping gene (upper panel). Cell viability was measured as in (A) (lower panel). ( C ) Venn diagram (left panel) and scatter plot (right panel) of the CTIP2-target genes ( , blue), the HMGA1-target genes ( , red) and the common subset (purple). The gene numbers of each set, the hypergeometric distribution P -value as well as the Pearson correlation coefficients for all three subsets and the fractions located in each quadrant of the scatter plot are indicated. ( D ) Overexpression of 7SK RNA in microglial cells. RNA amounts were monitored by ethidium bromide staining on agarose gel electrophoresis, and U6 RNA was used as a control. ( E ) Overexpression of a HA-tagged, dominant negative (dn) version of Cdk9 in microglial cells. dnCdk9 expression was monitored in western blot analyses using anti-HA and anti-Cdk9 antibodies. ( F ) as in (C), but for the target genes of dnCdk9 (yellow) and 7SK RNA overexpression (green). ( G ) shRNA-mediated knock-down of 7SK RNA in microglial cells. RNA amounts were monitored as in (D). Cell viability was measured as in (A). ( H ) Identification of 7SK-dependent P-TEFb targets as genes, whose expression is concomitantly regulated on dnCdk9 expression (yellow), 7SK RNA overexpression (green) and 7SK RNA knock-down (turquoise). The number of genes is plotted on the x-axis and the log 2 -fold change in each condition is plotted on the y-axis. Genes are plotted in descending order based on their expression changes on dnCdk9 expression. ( I ) Scatter plot comparing the expression changes of the P-TEFb targets identified in (H) on 7SK knock-down with their expression changes on CTIP2 knock-down. Genes statistically significantly regulated on CTIP2 knock-down are coloured in blue and the Pearson correlation coefficient is indicated. ( J ) Scatter plot comparing the expression changes of the P-TEFb targets identified in (H) on 7SK knock-down with their expression changes on HMGA1 knock-down. Genes statistically significantly regulated on HMGA1 knock-down are coloured in red.

    Journal: Nucleic Acids Research

    Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

    doi: 10.1093/nar/gku168

    Figure Lengend Snippet: HMGA1 and CTIP2 repress the expression of 7SK-dependent P-TEFb target genes. ( A ) shRNA-mediated knock-down of CTIP2 in microglial cells. CTIP2 levels were monitored in western blot analyses and β-actin was used as a control (upper panel). Cell viability on shRNA-mediated knock-down was measured by MTT test (lower panel). n.s., not statistically significant in a students t -test. ( B ) shRNA-mediated knock-down of HMGA1 in microglial cells. HMGA1 amounts were quantified by western blot analyses, and β-actin was used as a housekeeping gene (upper panel). Cell viability was measured as in (A) (lower panel). ( C ) Venn diagram (left panel) and scatter plot (right panel) of the CTIP2-target genes ( , blue), the HMGA1-target genes ( , red) and the common subset (purple). The gene numbers of each set, the hypergeometric distribution P -value as well as the Pearson correlation coefficients for all three subsets and the fractions located in each quadrant of the scatter plot are indicated. ( D ) Overexpression of 7SK RNA in microglial cells. RNA amounts were monitored by ethidium bromide staining on agarose gel electrophoresis, and U6 RNA was used as a control. ( E ) Overexpression of a HA-tagged, dominant negative (dn) version of Cdk9 in microglial cells. dnCdk9 expression was monitored in western blot analyses using anti-HA and anti-Cdk9 antibodies. ( F ) as in (C), but for the target genes of dnCdk9 (yellow) and 7SK RNA overexpression (green). ( G ) shRNA-mediated knock-down of 7SK RNA in microglial cells. RNA amounts were monitored as in (D). Cell viability was measured as in (A). ( H ) Identification of 7SK-dependent P-TEFb targets as genes, whose expression is concomitantly regulated on dnCdk9 expression (yellow), 7SK RNA overexpression (green) and 7SK RNA knock-down (turquoise). The number of genes is plotted on the x-axis and the log 2 -fold change in each condition is plotted on the y-axis. Genes are plotted in descending order based on their expression changes on dnCdk9 expression. ( I ) Scatter plot comparing the expression changes of the P-TEFb targets identified in (H) on 7SK knock-down with their expression changes on CTIP2 knock-down. Genes statistically significantly regulated on CTIP2 knock-down are coloured in blue and the Pearson correlation coefficient is indicated. ( J ) Scatter plot comparing the expression changes of the P-TEFb targets identified in (H) on 7SK knock-down with their expression changes on HMGA1 knock-down. Genes statistically significantly regulated on HMGA1 knock-down are coloured in red.

    Article Snippet: The primary antibodies used for ChIP were anti-CTIP2 (bethyl), anti-Cdk9 (Santa Cruz), anti-Cyclin T1 (Santa Cruz) and anti-Hexim-1 (Abcam).

    Techniques: Expressing, shRNA, Western Blot, MTT Assay, Over Expression, Staining, Agarose Gel Electrophoresis, Dominant Negative Mutation

    LSD1 cooperates with CTIP2 to repress HIV-1 gene transcription and viral replication. ( A ) Microglial cells were transfected with pNL-4.3 and the indicated plasmids (columns 2 to 4) or the pshRNA-control vector (column 1). Culture supernatants were analysed for p24 contents 48 h post-transfection. ( B and C ) Microglial cells were transfected with the episomal LTR-LUC and the indicated plasmids or the pshRNA-control vector. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with the episomal LTR-LUC and the control vectors (columns 1). DNA quantities were normalized with the pshRNA-control vector. ( D ) The knock-down efficiency of sh-RNA constructs was controlled by western blot analysis. The control columns 1 of the panels correspond to extracts from cells transfected with the pshRNA-control vector.

    Journal: Nucleic Acids Research

    Article Title: LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing

    doi: 10.1093/nar/gkr857

    Figure Lengend Snippet: LSD1 cooperates with CTIP2 to repress HIV-1 gene transcription and viral replication. ( A ) Microglial cells were transfected with pNL-4.3 and the indicated plasmids (columns 2 to 4) or the pshRNA-control vector (column 1). Culture supernatants were analysed for p24 contents 48 h post-transfection. ( B and C ) Microglial cells were transfected with the episomal LTR-LUC and the indicated plasmids or the pshRNA-control vector. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with the episomal LTR-LUC and the control vectors (columns 1). DNA quantities were normalized with the pshRNA-control vector. ( D ) The knock-down efficiency of sh-RNA constructs was controlled by western blot analysis. The control columns 1 of the panels correspond to extracts from cells transfected with the pshRNA-control vector.

    Article Snippet: LSD1 interacts with CTIP2 and co-localizes with Tat and CTIP2 in the nucleus Our data strongly suggest a functional cooperation between LSD1 and CTIP2.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Western Blot

    CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. ( A ) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. ( B ) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.

    Journal: Nucleic Acids Research

    Article Title: LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing

    doi: 10.1093/nar/gkr857

    Figure Lengend Snippet: CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. ( A ) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. ( B ) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.

    Article Snippet: LSD1 interacts with CTIP2 and co-localizes with Tat and CTIP2 in the nucleus Our data strongly suggest a functional cooperation between LSD1 and CTIP2.

    Techniques: Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Over Expression, Infection

    LSD1 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. ( A ) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. ( B–D ) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.

    Journal: Nucleic Acids Research

    Article Title: LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing

    doi: 10.1093/nar/gkr857

    Figure Lengend Snippet: LSD1 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. ( A ) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. ( B–D ) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.

    Article Snippet: LSD1 interacts with CTIP2 and co-localizes with Tat and CTIP2 in the nucleus Our data strongly suggest a functional cooperation between LSD1 and CTIP2.

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Immunodetection, Labeling, Staining

    COUP-TF-interacting protein 2 (CTIP2) impairs Vpr-mediated stimulation of p21 gene transcription and the resulting cell cycle arrest. ( a ) 293T cells inducible for HIV-1 Vpr expression, were transfected with the Flag-CTIP2 expression vector or the control

    Journal: Oncogene

    Article Title: p21WAF1 gene promoter is epigenetically silenced by CTIP2 and SUV39H1

    doi: 10.1038/onc.2009.193

    Figure Lengend Snippet: COUP-TF-interacting protein 2 (CTIP2) impairs Vpr-mediated stimulation of p21 gene transcription and the resulting cell cycle arrest. ( a ) 293T cells inducible for HIV-1 Vpr expression, were transfected with the Flag-CTIP2 expression vector or the control

    Article Snippet: Proteins were detected using antibodies directed against the FLAG epitope (M2 mouse monoclonal from Sigma, St Louis, MO, USA), CTIP2 (Santa Cruz, Santa Cruz, CA, USA), p21 (Santa Cruz), the HA epitope (Covance, Princeton, NJ, USA) and β-actin (Sigma).

    Techniques: Expressing, Transfection, Plasmid Preparation

    Trichostatin A (TSA) and chaetocin stimulate p21 gene transcription. ( a , b ) Microglial cells were transfected with the p21-LUC reporter plasmid in the presence or absence of pSuper-shRNA-CTIP2, as indicated. At 24-h post-transfection, cells were treated

    Journal: Oncogene

    Article Title: p21WAF1 gene promoter is epigenetically silenced by CTIP2 and SUV39H1

    doi: 10.1038/onc.2009.193

    Figure Lengend Snippet: Trichostatin A (TSA) and chaetocin stimulate p21 gene transcription. ( a , b ) Microglial cells were transfected with the p21-LUC reporter plasmid in the presence or absence of pSuper-shRNA-CTIP2, as indicated. At 24-h post-transfection, cells were treated

    Article Snippet: Proteins were detected using antibodies directed against the FLAG epitope (M2 mouse monoclonal from Sigma, St Louis, MO, USA), CTIP2 (Santa Cruz, Santa Cruz, CA, USA), p21 (Santa Cruz), the HA epitope (Covance, Princeton, NJ, USA) and β-actin (Sigma).

    Techniques: Transfection, Plasmid Preparation, shRNA

    COUP-TF-interacting protein 2 (CTIP2) relocates Vpr to subnuclear structures. Microglial cells were transfected with pVpr-GFP alone (panels 1, 2 and 3) or together with pRFP-CTIP2 plasmid (panels 4–10). Cells were fixed 24-h post-transfection

    Journal: Oncogene

    Article Title: p21WAF1 gene promoter is epigenetically silenced by CTIP2 and SUV39H1

    doi: 10.1038/onc.2009.193

    Figure Lengend Snippet: COUP-TF-interacting protein 2 (CTIP2) relocates Vpr to subnuclear structures. Microglial cells were transfected with pVpr-GFP alone (panels 1, 2 and 3) or together with pRFP-CTIP2 plasmid (panels 4–10). Cells were fixed 24-h post-transfection

    Article Snippet: Proteins were detected using antibodies directed against the FLAG epitope (M2 mouse monoclonal from Sigma, St Louis, MO, USA), CTIP2 (Santa Cruz, Santa Cruz, CA, USA), p21 (Santa Cruz), the HA epitope (Covance, Princeton, NJ, USA) and β-actin (Sigma).

    Techniques: Transfection, Plasmid Preparation