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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. 5 — β3-AR blockade reduces stemness in human neuroblastoma BE(2)C cells by modulating the SK2/S1P₂ signaling axis. Neurospheres formation assay of human neuroblastoma BE(2)C cell line. Cells were plated in MW24 (1 × 10⁴ cells/well) with Neurosphere basic medium (see “Methods” section) and after 24 h treated with 1 μM SR59230A or 1 μM BRL37344, in presence or not of 10 μM CYM5520 or 1 μM ABC294640. Once formed, spheres were disrupted and cells re-plated for a second passage (P2). a Images were taken after 7 days of neurosphere formation (P2). The effect of the treatments on stemness features of BE(2)C neurospheres, was evaluated by using different assays. b Number of neurospheres formed per well was counted using an optical microscope and compared to the control condition. Results are reported as mean ± SD and are representative of three independent experiments. Significance was calculated by Unpaired t-test analysis with equal SD (**P < 0.01, ***P < 0.001) and two-way ANOVA analysis followed by Bonferroni’s post hoc test (^§§§P < 0.001 ABC+BRL vs. BRL). c Average diameter of the spheres was quantified by using the ImageJ Software. Results are reported as mean ± SD and are representative of three independent experiments. Significance was calculated by Unpaired t-test analysis with equal SD (**P < 0.01, ***P < 0.001) and two-way ANOVA analysis followed by Bonferroni’s post hoc test (^#P < 0.05 CYM+SR vs. SR; ^§§§P < 0.001 ABC+BRL vs. BRL). d Flow cytometry analysis of neurospheres was performed by using MACSQuant Analyzer 10 (Miltenyi Biotech). After disruption of the spheres, cells were stained with anti-CD34-PE-Vio770, anti-CD133-APC antibodies and Viobility Fixable dyes. Results were reported as mean ± SD of CD34 positive cells and are representative of three independent experiments. Significance was calculated by Unpaired t-test analysis with equal SD (**P < 0.01, ***P < 0.001) and two-way ANOVA analysis followed by Bonferroni’s post hoc test (^###P < 0.001 CYM+SR vs. SR; ^§§P < 0.01 ABC+BRL vs. BRL)