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Additional Supporting Information may be found in the online version of this article.

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STEM_541_sm_suppinfofigureandtables.pdf6281KSupplemental Table S1. Genes showing differential expression between Nes −/− and wildtype NSCs. Figure S1. GFAP staining in forebrain sections of E11.5 embryos. Scale bars equal 50 μm. Figure S2. BrdU staining in hindbrain sections of E11.5 embryos (A) and in cultured NSCs derived from E11.5 embryos (C), as well as quantitation of BrdU labeling in vivo (B) and in vitro (D). Nuclei were counterstained with hematoxylin. Scale bars equal 100 μm. Figure S3. SOX2 (green) and Ki67 (red) co-staining in forebrain sections of E11.5 embryos (A) and quantitation of stained cells (B). Scale bars equal 100 μm. Figure S4. Increased apoptosis in Nes−/− embryos based on aCASP3 staining. Sections from the forebrain of E10.5 (left), E11.5 (middle) and E12.5 (right) embryos were immunostained for aCASP3 (red) and counterstained with DAPI (blue). Scale bar equals 100 μM. Figure S5. SOX2 and aCASP3 co-staining in forebrain sections of E11.5 embryos (A) and quantitation of stained cells (B). Scale bars equal 100 μm. Figure S6. Disruption of microtubules by colchicine. NSCs were treated with varying concentrations of colchicine. Cells were then stained with anti-α-tubulin to visualize microtubules (green) and rhodamineconjugated phalloidin to visualize actin microfilaments (red). Microtubules were slightly affected at 0.2 μM and severely disrupted at 1 μM of colchicine, with comparable effects observed between Nes+/+ and Nes−/− cells. When microtubules were disrupted by 1 μM of colchicine, actin polymerization was increased in both genotypes. Scale bar equals 50 μm. Figure S7. Disturbance of actin microfilaments by cytochalasin D. NSCs were treated with varyingconcentrations of cytochalasin D. Cells were then stained with rhodamine-conjugated phalloidin to visualize actin microfilaments. Microfilaments were slightly affected at 20nM and noticeably disturbed 200 nM of cytochalasin D, with comparable effects observed between Nes+/+ and Nes−/− cells. Scale bar equals 50 μm. Figure S8. Effects of CDK inhibition on the apoptosis phenotype of Nes−/− NSCs. Cells were treated with roscovitine (A) or 3-amino-1H pyrazolo[3,4-b]quinoxaline (B) at varying concentrations followed by the measurement of caspase-3/7 activity. Error bars represent SE from four independent experiments. Asterisks indicate cases where drug-treated Nes−/− cells exhibit a statistically significant reduction in caspase-3/7 activity as compared to Nes−/− cells w drug treatment.

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