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ANA_22593_sm_SuppFig1.tif6105KSupplementary Fig S1. Time-course of myelination in neonatal mouse forebrain slices. (A) Coronal brain slices from P2 mice were cultured for 1, 2, 3, 4, 7, 10 days in vitro (div) then immunostained with anti-MBP (green) and with anti-NF (red) antibodies. The presence of the first myelinated bundles was seen in the deepest layer of the cingulate cortex and the cingulum at 4 div, region denoted by dotted boxes. Myelination process was more extensive at 10 div. Scale bar: 50 μm. (B) Higher magnification images represent the boxed region in the upper panel in A. At 1 to 3 div we observed a progressive increase of the number of MBP-positive cells. By 4 div many axons were myelinated in the cingulate cortex then from 7 to 10 div myelination spread to the entire corpus callosum and cortex. Scale bar: 25 μm.
ANA_22593_sm_SuppFig2.tif854KSupplementary Fig S2. Schematic representation of the experimental protocol in healthy (A) neonate or (B) adult animals. (n) Number of animals treated in each case. Animals were sacrificed at different time points for immunohistological (Histo) or electron microscopy (EM) analysis.
ANA_22593_sm_SuppFig3.tif259KSupplementary Fig S3. Schematic representation of the experimental protocol in mouse models of demyelination. (A-B) LPC or (C) cuprizone models. Animals were sacrificed at different time points for immunohistological (Histo) or electron microscopy (EM) analysis. In B the same animals were analyzed at both time points by MRI. (n) Number of animals treated in each case.
ANA_22593_sm_SuppFig4.tif3501KSupplementary Fig S4. Typical axial T2-weighted MRI of vehicle-treated or olesoxime-treated mice injected with LPC. BI, before LPC injection; W1, 7 days after LPC injection; W2, 14 days after LPC injection. Arrows point at the lesion induced by LPC in the corpus callosum.
ANA_22593_sm_SuppTab1.doc31KSupplementary Table S1. Effect of olesoxime on OPC survival and proliferation in organotypic slice culture. Brain sections were maintained in the presence of olesoxime for 4 days (as described in figure 1) and BrdU was added to the culture media 12h before fixation. Caspase analysis was performed at the same time point. Values shown are means ± SEM (n=3). There was no statistically significant effect of olesoxime on cell survival or proliferation in this in vitro system.
ANA_22593_sm_SuppTab2.doc30KSupplementary Table S2. Effect of olesoxime on cell survival and proliferation in newborn mice. Neonatal mice were fed for 2 weeks with olesoxime and injected with BrdU 24 hours before sacrifice. The number of proliferating OPCs in the corpus callosum remained unchanged and in addition, there was no difference in the number of apoptotic cells. Values shown are means ± SEM (n=5). There was no statistically significant effect of olesoxime on cell survival or proliferation in vivo during development.
ANA_22593_sm_SuppTab3.doc31KSupplementary Table S3. Effect of olesoxime on cell survival and proliferation in adult mice. Adult mice were treated for 2 weeks with olesoxime and BrdU was administrated in drinking water for the last 5 days before sacrifice. We quantified cycling cells in the SVZ (PH3+ cells), BrdU+ cells and apoptotic caspase3+ cells in the corpus callosum and found no differences in olesoxime-treated animals compared to controls. Values shown are means ± SEM (n=3). There was no statistically significant effect of olesoxime on cell survival or proliferation.
ANA_22593_sm_SuppTab4.doc31KSupplementary Table S4. Effect of olesoxime on cell survival and proliferation in the LPC mouse model. We quantified 7 days after LPC injection, cycling progenitors (PH3+/PDGFRa+ cells) and dying cells (Caspase3+ cells) at the lesion site and proliferating progenitors within the SVZ (PH3+ cells). We found no differences in olesoxime-treated animals compared to controls at the two doses. Values shown are means ± SEM (n=3).
ANA_22593_sm_SuppTab5.doc31KSupporting Information Table 5. Supplementary Table S5. Effect of olesoxime on cell proliferation in the cuprizone mouse model. We quantified cycling cells in demyelinated the CC (PH3+ cells) and found no differences in olesoxime-treated animals compared to controls at the W5 and W7. We observed a 37% but non-significant increase in the number of proliferating cells in the corpus callosum of olesoxime treated animals. This effect in proliferation is not observed anymore at W7. This result could indicate that olesoxime has a transient effect on progenitor proliferation leading to a transient increase in the number of Olig2+cells in the CC of treated animal. Values shown are means ± SEM (n=4 at W5 and n=3 at W7).

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