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. 2019 Mar 14;9:4572. doi: 10.1038/s41598-019-41040-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of conditioned media obtained from mouse NP-derived astrocyte-like cells on neuronal cell number and cell death. Non transgenic mNPsc were pre-differentiated for 10 days and then exposed for additional five days to CMs obtained from astrocyte-like cells (CM + Dox + IL1β or CM − Dox + IL1β). (A) Representative microphotographic fields showing neuronal cells stained with an anti-MAP2 antibody. Scale bar 30 μm. (B) Neuronal and activated Caspase-3 positive cell quantification. The percentage of MAP2 positive neuronal cells was reduced in cultures treated with CM + Dox + IL1β compared with CM − Dox + IL1β treated cultures; data are means ± SEM, n = 3, *P < 0.005 (paired Student’s T test). Immunocytochemical detection of activated Caspase-3 showed that CM + Dox + IL1β increased cell death compared to CM − Dox + IL1β after three days of treatment. Quantification of Caspase-3 stained cells is given in the bar graph, and expressed with respect to CM − Dox + IL1β; data are means ± SEM, n = 3, *P < 0.0005. (C) Representative image showing cells immunostained for βIII tubulin (green) and Casp-3 (red). The arrowhead indicates a neuronal cell expressing activated Casp-3. Note also the presence of Casp-3 positive cells not expressing βIII tubulin (arrows). Scale bar 30 μm).