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Fig. S1. Deglycanation affects neurosphere size distribution and total cell number. (A) Quantitative analysis of Nsph sizes that were arbitrarily divided into four classes according to Nsph diameter, as indicated. Note that the treatment with ChABC (light gray) causes a reduction in Nsph size in comparison with untreated sibling cultures (dark gray), which is reflected in significantly higher numbers of small Nsphs and diminished numbers of large Nsphs. (B) Accordingly, the total cell number obtained from ChABC-treated cortical and striatal Nsphs is significantly reduced. The statistical significance according to Student’s t-test is indicated: *, P<0.05; **, P<0.01; ***, P<0.001; n=3 independent experiments were carried out.
Fig. S2. The neurosphere differentiation assay reveals the functional relevance of CS-GAGs. (A) Photomicrographs of cultures developed as indicated on each panel. The efficient removal of CS-GAGs from differentiating Nsph cultures by ChABC treatment was controlled by immunocytochemistry after 3 div. Nsph-derived cells cultivated under control conditions (w/o, upper panels), in the presence of 50 mU ChABC (middle panels) or keratanase (lower panels) were analyzed by immunostaining using antibodies 473HD and pk-anti-phosphacan (KAF13), which detect a unique CS-GAG and the core proteins of the Rptpβ gene products, respectively. Note the strong expression of the 473HD epitope on individual cells under control and keratanase conditions, which was colocalized with KAF13 staining of DSD-1-PG/RPTP-β core proteins and absent after ChABC-treatment. Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). (B,C) For quantitative analysis, the proportion of immunopositive cells in the differentiation assay was determined and registered as percentage fraction from the total population, as assessed from nuclear staining. The relative numbers of neural cell types occurring in cortical (B) or striatal (C) Nsphs after 6 div is plotted. Note that ChABC-treatment (gray) selectively caused a significant decrease in the number of nestin-positive precursor cells and βIII-tubulin-positive neurons in comparison with control (dark gray) and keratanase-treated cultures (light gray). This reduction was accompanied by a concomitant increase in the number of GFAP-positive astrocytes. Equivalent results were obtained in differentiation assays using cortical (B) and striatal (C) Nsphs. Scale bar: 20 μm.
Fig. S3. The TUNEL reaction revealed no difference in cell apoptosis between control and ChABC-injected groups. For visualization of the apoptotic cell rate in the developing forebrain 1 day after intracerebroventricular (ICVI) injection of ACSF and ChABC in utero, we used fluorescein-dUTP to label DNA strand breaks that are generated during apoptosis (TUNEL reaction). Only very few cells dying by apoptosis were detectable in TUNEL stainings, and no differences between ChABC-injected (middle panels) and ACSF-injected (upper panels) embryos could be discerned. The positive control for the TUNEL reaction was performed on DNaseI-treated sections and was included in each experimental set (lower panel). Scale bar: 100 μm.
Fig. S4. Deglycanation does not affect the integrity of adherens junction components. Photomicrographs of double-immunostained frontal forebrain sections of E14.5 embryos depicting the expression of E-cadherin, aPKC and laminin (all in green) in comparison with the 473HD epitope (red). Note that there is no apparent change in the pattern of immunoreactivity observed between ChABC-injected (right panels) and ACSF-injected (left panels) embryos, although, as expected, the 473HD immunoreactivity is lost in the ChABC-injected embryos. This indicates that the treatment did not modify the cell polarity. Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). Scale bar: 50 μm.
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