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1.
FIG. 6.

FIG. 6. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

LSD1 inhibitor treatment leads to reduced cell proliferation in the dentate gyri of adult mouse brains. (a) Representative images of hippocampal dentate gyrus brain sections of wild-type adult mice that were injected with saline, pargyline (Par) or tranylcypromine (Tranyl) and treated with BrdU. Brain sections were stained with BrdU (green) to measure cell proliferation. NeuN staining (red) was included to show the structure of dentate gyrus. (b) Average numbers of BrdU-positive (BrdU+) cells in the subgranular zone (SGZ) of the dentate gyrus (DG) in one field of 20-μm wild-type brain sections. (n = 6 mice for each treatment group). S, saline; P, pargyline; T, tranylcypromine. Error bars are standard deviations of the means. *, P < 0.001 by one-way ANOVA. (c) Representative images of hippocampal dentate gyrus brain sections of TLX−/− adult mice that were injected with saline, pargyline (Par), or tranylcypromine (Tranyl) and treated with BrdU. Brain sections were stained with BrdU (green) and NeuN (red). The BrdU-positive cells that are located in the SGZ of DG are indicated by arrows. (d) Average numbers of BrdU-positive (BrdU+) cells in the SGZ of the DG in one field of 20-μm TLX−/− brain section. (n = 6 mice for each treatment group). S, saline; P, pargyline; T, tranylcypromine. Error bars are standard deviations of the means.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.
2.
FIG. 1.

FIG. 1. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

LSD1 is expressed in neural stem cells and interacts with TLX. (a) Expression of LSD1 in neural stem cells revealed by immunofluorescence analysis. LSD1 staining is shown in red, and DAPI (4′,6′-diamidino-2-phenylindole) staining is shown in blue. The merged image of LSD1 staining, DAPI staining, and phase-contrast image (gray) is shown at the bottom panel. (b) Expression of LSD1 in neural stem cells cultured under proliferation (P) or differentiation (D) conditions. The levels of monomethyl H3K4 (1me-H3K4) and dimethyl H3K4 (2me-H3K4) are shown in parallel. Total histone H3 levels were included as a control. GAPDH was included as a loading control. (c) Expression of TLX in neural stem cells cultured under proliferation (P) or differentiation (D) conditions. GAPDH was included as a loading control. (d) TLX interacts with LSD1 in HEK 293 cells as revealed by reciprocal coimmunoprecipitation analysis. Lysates of HA-TLX- and Flag-LSD1-transfected cells were immunoprecipitated with anti-HA or anti-Flag antibody, followed by immunoblotting with anti-Flag or anti-HA antibody. Protein expression in cell lysates was shown by immunoblotting with anti-HA or anti-Flag antibody. IP, immunoprecipitation; IB, immunoblotting. (e) TLX interacts with LSD1 and HDAC5 in neural stem cells, analyzed by immunoprecipitation analysis. Lysates of neural stem cells were immunoprecipitated with TLX-specific antibody (αTLX), followed by immunoblotting with anti-LSD1 or anti-HDAC5 antibody. IgG was included as a negative control. Ten percent input was loaded on the gel and included as a control.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.
3.
FIG. 3.

FIG. 3. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

Knockdown of LSD1 expression leads to induced p21 and pten gene expression and reduced neural stem cell proliferation. (a and b) LSD1 siRNA treatment leads to increased mono- and dimethyl H3K4 (1me-H3K4 and 2me-H3K4) levels on p21 (a) and pten (b) promoters in neural stem cells as analyzed by quantitative ChIP assays. Input, DNA input; IgG, IgG control. Antibodies specific for TLX, LSD1, 1me-H3K4, 2me-H3K4, and 2me-H3K9 were included in the assay. (c) Gene expression regulated by LSD1-specific siRNA treatment as revealed by RT-PCR analysis. Neural stem cells were treated with control siRNA (C), LSD1 siRNA (siLSD1), TLX siRNA (siTLX), or the combination of both (siTLX/siLSD1). Actin was included as a loading control. (d) LSD1 siRNA reduces neural stem cell proliferation as revealed by decreased BrdU labeling (red). Neural stem cells were transfected with control siRNA (C, panels 1), LSD1 siRNA (siLSD1, panels 2), TLX siRNA (siTLX, panels 3), or the combination of LSD1 siRNA and TLX siRNA (siTLX+siLSD1, panels 4). (e) Percentages of BrdU-positive (BrdU+) cells in control (bar 1)-, LSD1 siRNA (bar 2)-, TLX siRNA (bar 3)-, and TLX siRNA plus LSD1 siRNA (bar 4)-treated neural stem cells. Error bars are standard deviations of the means; assays were repeated three times. *, P < 0.001 by one-way ANOVA.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.
4.
FIG. 4.

FIG. 4. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

Simultaneous knockdown of LSD1 and HDAC5 led to dramatic inhibition of neural stem cell proliferation. (a) TLX recruitment of LSD1 and HDAC5 to the promoters of p21 and pten genes, analyzed by ChIP assays. Input, DNA input; IgG was included as a negative control. (b) The knockdown effect of the LSD1 siRNA (siLSD1) and the HDAC5 siRNA (siH5) in neural stem cells was analyzed by semiquantitative RT-PCR. Actin was included as a loading control. (c) Induction of p21 and pten gene expression in neural stem cells treated with LSD1 siRNA (siLSD1) or HDAC5 siRNA (siH5) or the combination of both (siLSD1/siH5) as analyzed by quantitative real-time RT-PCR. The expression levels of p21 and pten were normalized by actin expression levels and plotted. (d) Treatment of LSD1 siRNA and/or HDAC5 siRNA reduces neural stem cell proliferation. Cell proliferation was analyzed by BrdU labeling (red). Nuclear DAPI staining is shown in blue. Neural stem cells were transfected with control siRNA (C, panel 1), HDAC5 siRNA (siHDAC5, panel 2), LSD1 siRNA (siLSD1, panel 3), or the combination of LSD1 and HDAC5 siRNA (siLSD1+siHDAC5, panel 4). (e) Percentages of BrdU-positive cells in control (bar 1)-, HDAC5 siRNA (bar 2)-, LSD1 siRNA (bar 3)-, and LSD1 siRNA+ HDAC5 siRNA (bar 4)-treated neural stem cells. Error bars are standard deviations of the mean; assays were repeated three times. *, P < 0.01 by one-way ANOVA.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.
5.
FIG. 5.

FIG. 5. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

LSD1 siRNA treatment leads to reduced cell proliferation in the dentate gyri of adult mouse brains. (a) Expression of LSD1 in the subgranular zone (SGZ) of the dentate gyri of wild-type adult mouse brains as revealed by immunostaining. The LSD1 staining is shown in red. Cells indicated by arrows are examples of the LSD1-positive cells in the SGZ. An enlarged image of the LSD1-positive cells in the boxed region is shown on the right. (b) Knockdown of LSD1 expression using the LSD1 siRNA-expressing lentivirus in cultured neural stem cells. SC, scrambled siRNA control. Actin was included as a loading control. (c) Reduced BrdU staining of LSD1 siRNA-transduced cells in the dentate gyri (DG) of adult brains. DG sections from scrambled siRNA control- or LSD1 siRNA-transduced and BrdU-treated mice were immunostained for BrdU (blue). NeuN staining (red) was included to show the structure of the DG. The virus-transduced cells are shown in green due to the expression of a GFP marker. (d) Percentages of doubly GFP-positive and BrdU-positive cells out of GFP+ cells in the dentate gyrus SGZ of the LSD1 siRNA lentivirus-infected mice. SC, scrambled control siRNA; siLSD1, LSD1 siRNA. Data are represented as means ± standard deviations. *, P < 0.01 by Student's t test. (e) Examples of scrambled siRNA control (sc siRNA-GFP)- or LSD1 siRNA (siLSD1-GFP)-transduced cells in the dentate gyrus (DG) SGZ. LSD1 staining is shown in red. The virus-transduced cells are shown in green due to the expression of a GFP marker. The merged images are shown on the right.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.
6.
FIG. 2.

FIG. 2. From: Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation .

LSD1 inhibitor treatment inhibits neural stem cell proliferation. (a) Cell proliferation was revealed by BrdU labeling (red) in neural stem cells treated with different concentrations (0, 1.2, and 2.4 mM) of pargyline. The images shown are BrdU staining merged with phase contrast images. (b) Percentage of BrdU-positive (BrdU+) cells in 0, 1.2 mM, and 2.4 mM pargyline-treated neural stem cells. Error bars are standard deviations of the means; assays were repeated three times. *, P < 0.01; **, P < 0.001 (by Student's t test). (c) Cell proliferation was revealed by BrdU labeling (red) in solvent (0 μM)- and tranylcypromine (2 μM)-treated neural stem cells. (d) Percentage of BrdU-positive cells in solvent- and tranylcyptomine-treated neural stem cells. Error bars are standard deviations of the means; assays were repeated three times. *, P < 0.001 by Student's t test. (e) Gene expression regulated by the LSD1 inhibitor pargyline, revealed by RT-PCR analysis. Actin was included as a loading control. (f) Gene expression regulated by the LSD1 inhibitor tranylcypromine, revealed by RT-PCR analysis. (g) Pargyline and tranylcypromine treatment relieve TLX-mediated repression of p21 promoter-driven luciferase (p21-luc) activity. CV-1 cells were transfected with p21-luc along with the LSD1 expression vector and a control vector (−TLX) or with the LSD1 expression vector and the TLX-expressing vector (+TLX). The transfected cells were treated with solvent (C), pargyline (P), or tranylcypromine (T). Fold repression was determined by dividing luciferase activity in TLX-transfected cells (+TLX) with luciferase activity in control vector-transfected cells (−TLX) for each treatment. *, P < 0.01 by Student's t test. (h and i) Pargyline and tranylcypromine treatment lead to increased mono- and dimethyl H3K4 (1me-H3K4 and 2me-H3K4) levels on p21 (h) and pten (i) promoters in neural stem cells, analyzed by quantitative ChIP assays. Input, DNA input; IgG was included as a negative control. Antibodies specific for TLX, LSD1, 1me-H3K4, 2me-H3K4, and 2me-H3K9 were included in the assay. C, solvent control; P, pargyline; T, tranylcypromine.

GuoQiang Sun, et al. Mol Cell Biol. 2010 April;30(8):1997-2005.

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