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. 2019 Sep 2;39(2):368–384. doi: 10.1038/s41388-019-0993-1

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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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Fig. 7 — Neuroblastoma tumor growth reduction exerted by SR59230A via SK2/S1P₂ signaling axis is accompanied with an increased neuronal differentiation of NB cells. a WB and relative densitometric quantification analysis, showing the protein expression levels of the early differentiation marker NeuroD1. Lysates were obtained from tumor mass (at 14 days) of Vehicle-, SR59230A-, CYM5520- and SR59230A+CYM5520-treated mice (n = 3 for each group) (top) and Vehicle-, SR59230A-, ABC294640- and SR59230A+ABC294640-treated mice (n = 3 for each group) (bottom). Significance was calculated by two-way ANOVA analysis followed by Bonferroni’s post hoc test (*P < 0.05, **P < 0.01; ^#P < 0.05 SR+CYM vs. SR). b Upper panel: Immunofluorescence of paraffin-embedded tumor sections showing expression of the neuronal differentiation marker Map2. Images are representative of similar results obtained for Vehicle- (n = 5), SR59230A- (n = 5), CYM5520- (n = 5), SR59230A+CYM5520- (n = 5), ABC294640- (n = 5) and SR59230A+ABC294640-treated mice (n = 5). Lower panel: Quantification of Map2-associated fluorescence intensity. Data are mean ± SD of eight fields of each specimen quantified in three independent experiments. Significance was calculated by one-way ANOVA analysis followed by Bonferroni’s post hoc test (****P < 0.0001 SR vs. Vehicle, ***P < 0.0001 ABC vs. Vehicle, ****P < 0.0001 SR+ABC vs. Vehicle; ^####P < 0.0001 SR+CYM vs. SR. c Upper panel: Immunofluorescence of paraffin-embedded tumor sections showing expression of the neuronal differentiation marker Neurofilament. Images are representative of similar results obtained for Vehicle- (n = 5), SR59230A- (n = 5), CYM5520- (n = 5), SR59230A+CYM5520- (n = 5), ABC294640- (n = 5) and SR59230A+ABC294640-treated mice (n = 5). Lower panel: Quantification of Neurofilament-associated fluorescence intensity. Data are mean ± SD of eight fields of each specimen quantified in three independent experiments. Significance was calculated by one-way ANOVA analysis followed by Bonferroni’s post hoc test (****P < 0.0001 SR vs. Vehicle, ***P < 0.0001 ABC vs. Vehicle, ****P < 0.0001 SR+ABC vs. Vehicle; ^####P < 0.0001 SR+CYM vs. SR