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STEM_1405_sm_SupplFigure1.tif132KFigure S1. ZAC1 transactivates the human SOCS3 gene. (A): Scheme of the SOCS3 5′end including the promoter, exons (filled boxes) and first intron. Signal flag symbolizes transcriptional start site. Predicted ZAC1 DNA-binding sites are depicted by filled bars and circles. ChIP primer pairs flanking amplified regions are shown by filled triangles and Roman numbers, respectively. (B): Reporter assays in SK-N-MC cells cotransfected with increasing amounts of human ZAC1 (50 ng, 100 ng, 250 ng, 500 ng) and a 1.7 kb human SOCS3 promoter construct (200 ng). (C): Transfection of ZAC1 (200 ng) increased SOCS3 expression compared to mock transfection as determined by qRT-PCR. (D): Immunoblot (WCE 50 μg) analysis of ZAC1 and SOCS3 expression following mock and ZAC1 (200 ng) transfection (E): ChIP assay with a ZAC1 antibody showed binding at the SOCS3 promoter (II), exon 1 (III) and intron (IV) in mock and ZAC1 (200 ng) transfected SK-N-MC cells. No binding could be detected at the upstream region (I) devoid of ZAC1 DNA binding motifs. Representative results from 3 (D) or means with standard deviations (± SD) from 6 independent (B, C, E) experiments are shown. * P < 0.05 and ** P < 0.01.
STEM_1405_sm_SupplFigure2.tif223KFigure S2. Zac1 induces Socs3 expression during astroglial differentiation of NS-5 cells. (A): Promoter reporter assays. Cotransfection of increasing amounts of Zac1 (5 ng, 25 ng, 50 ng, 100 ng) enhanced Socs3 promoter (200 ng) activity. (B): Astroglial and neuronal differentiation of the embryonic NSC line NS-5. Expression of Nestin, Gfap and Tuj1 was monitored by RT-PCR. (C): Time course analysis of Zac1 and Socs3 mRNA expression during astroglial and neuronal cell differentiation as measured by qRT-PCR. Zac1 was expressed in both lineages, whereas Socs3 induction was confined to the astroglial lineage. (D): Immunoblot analysis (WCE 70 μg) revealed concomitant increases in Zac1 and Socs3 expression during astroglial differentiation, while Zac1 expression during neuronal differentiation did not enhance Socs3 expression. (E): ChIP analysis during astroglial or neuronal differentiation evidenced Zac1 occupancy at the Socs3 promoter, exon 1 and intron solely during the former. Representative results from 3 (B, D) or means ± SD from 6-7 independent (A, C, E) experiments are shown. * P < 0.05 and ** P < 0.01.
STEM_1405_sm_SupplFigure3.tif170KFigure S3. Histone marks at the Socs3 gene during neuronal differentiation of O4ANS cells. (A and B): Chromatin marks during neuronal differentiation at day 4 were detected with antibodies against acH3 (A) and H3K9me2 (B). (C and D): Sequential ChIP analysis during neuronal differentiation showed unchanged association of Zac1 with active (acH3/Zac1) (C) or repressive (H3K9me2/Zac1) (D) chromatin marks. Means ± SD from 4 independent experiments are shown.
STEM_1405_sm_SupplFigure4.tif37KFigure S4. Activated polymerase II is recruited during astroglial differentiation. ChIP assay against activated polymerase II (Ser5-Pol) were performed on day 4 in differentiating O4ANS cells. Activated polymerase II is recruited during astroglial, but not neuronal, differentiation. Means ± SD from 4 independent experiments are shown. * P < 0.05 and ** P < 0.01.
STEM_1405_sm_SupplFigure5.tif115KFigure S5. Demethylation of Socs3 during astroglial differentiation (A): Scheme of Socs3 upstream region with overlaying CpG island shown beneath. Primer pairs flanking amplified regions are indicated. (B): MeDIP assay of the upstream region evidenced low methylation at the promoter and transcriptional start site, whereas higher levels of methylation were detected at the exonic and intronic regions. In either case reduced DNA methylation at different Zac1 binding sites preferentially occurred during astroglial differentiation. (C): Overall methylation at the Socs3 upstream region following astroglial and neuronal differentiation. Means ± SD from 4 independent (B, C) experiments are shown. * P < 0.05 and ** P < 0.01.
STEM_1405_sm_SupplFigure6.tif429KFigure S6. Zac1 expression inhibits Stat3 (Y705) phosphorylation (A): Immunoblot (WCE 70 μg) of mock or Zac1 (250 ng) transfected C17.2 cells. Zac1 transfection increased Socs3 expression and decreased Stat3 (Y705) phosphorylation while expression of Stat3 and β-actin proteins was unaffected. (B): Following astroglial differentiation of O4ANS, Stat3 (Y705) phosphoimmunoreactivity decreased as evidenced by immunoblot (WCE 70 μg). In contrast, Stat3 protein expression was unaltered under either astroglial or neuronal differentiation. Representative results from 3 independent experiments are shown.
STEM_1405_sm_SupplFigure7.tif2724KFigure S7. Glial markers colocalize with Zac1 or Socs3 during the transition to differentiation. (A): O4ANS undergoing astroglial differentiation were subjected to indirect immunofluorescence with antibodies A2B5 (red) (a-j), Gfap (green) (k-t), Zac1 (green) (a-e) and (red) (k-o), Socs3 (green) (f-j) and (red) (p-t). Nuclei were stained with DAPI (blue). Scale bar 40 μm. Representative results from 5 independent experiments are shown.
STEM_1405_sm_SupplFigure8.tif2630KFigure S8. Zac1 induces Socs3 in differentiating astroglial cells during the transition to maturation. (A): Astroglial differentiation of NS-5 cells. At different days cells were subjected to indirect immunofluorescence with antibodies against A2B5 (a-d) and Gfap (e-h), merge (i-l) or Zac1 (a′-d′) and Socs3 (e′-h′), merge; (i′-l′). Nuclei were stained with DAPI (blue). Scale bar 40 μm. (B): Quantitative analysis of A2B5 and Gfap positive cells are shown as percentage of the total number of DAPI positive cells. Mean of five analyzed images per day of the type shown in (A, a-l). (C): Quantitative analysis of Zac1 and Socs3 positive cells are shown as percentage of the total number of DAPI positive cells. Mean of five analyzed images per day of the type shown in (A, a′-l′). (D): Colocalization of Zac1 (green) (a-d) and (red) (i-l), and Socs3 (green) (e-h) and (red) (m-p) with glial markers A2B5 (red) (a-h) or Gfap (green) (i-p). Scale bar 40 μm. Representative results from 5 independent experiments are shown.
STEM_1405_sm_SupplFigure9.tif1145KFigure S9. Socs3 knock-down reinstates astroglial differentiation in Zac1 overexpressing O4ANS cells. (A): Zac1 and Socs3 expression in parental, Flag-Zac1 (FZ) overexpressing, and Flag-Zac1 overexpressing Socs-3 knock-down (FZ-shRNA-Socs3) cells were measured by qRT-PCR whereby parental expression was set to 100%. (B): Integration of Socs3 shRNA plasmids was evidenced by the selection marker neomycin. (C): Zac1 and Socs3 expression in FZ-shRNA-Socs3 cells during astroglial differentiation as measured by qRT-PCR. (D): Distribution of cell cycle phases in FZ and FZ-shRNA-Socs3 cells during astroglial differentiation. (E): Immunocytochemistry of FZ and FZ-shRNA-Socs3 cells during astroglial differentiation. Cells were subjected to indirect immunofluorescence with antibodies against A2B5 (red) and Gfap (green). Nuclei were stained with DAPI (blue). Scale bar 40 μm. Representative results from 2-4 (B, E) or means ± SD from 6-8 independent (A, C, D) experiments are shown. * P < 0.05 and ** P < 0.01.
STEM_1405_sm_SupplTable1.pdf22KTable S1. Primers for PCR and ChIP experiments

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