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. 2016 Sep 22;7(9):e2376. doi: 10.1038/cddis.2016.195

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Copyright © 2016 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Compound 12 inhibition of Hh-dependent MB tumor cell growth. (a–c) Ex vivo cell cultures from Ptch1^+/− mice MBs were treated with compound 12, Cyclopamine, Vismodegib, LDE-225 or DMSO only. (a and b) After the indicated times, a trypan blue count was performed to determine the growth rate of viable cells. (c) Gli1 mRNA expression levels were determined by qRT-PCR normalized to endogenous control (β2-microglobulin and HPRT). (d–f) Compound 12 inhibits MB-SCs self-renewal. (d) Suspension of single MB-SCs isolated from Ptch1^+/− mice were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of compound 12, Cyclopamine or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self-renewal MB-SCs' capability is expressed as percentage of neurosphere-forming cells. (e) Representative bright-field images of tumor neurospheres after compound 12 or Cyclopamine treatment are also shown. (f) MB-SCs isolated from Ptch1^+/− mice MBs were treated for 48 h with compound 12 or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA. For qRT-PCR, results were normalized to endogenous control (β2-microglobulin and HPRT). All data show the mean±S.D. of three independent experiments. *P<0.05; **P<0.01 versus DMSO (CTR)