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. 2022 Mar 4;59(5):2932–2945. doi: 10.1007/s12035-022-02771-0

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Mibefradil induces cell death in MB spheres. A Left panel: representative bright field and fluorescent images of CHLA-01R cell spheres at before (day 5) and after (day 8) treatment with mibefradil (10 µM) or DMSO control. The scale bar is 300 μm. Right panel: quantitative analysis of PI fluorescence intensity of DMSO control and increasing concentrations of mibefradil. Results for each concentration were standardised to the non-treated control and expressed as fold change. Data represents mean ± standard deviation from three independent experiments with three replicates each. ns, not significant (p > 0.05), *p < 0.01, **p < 0.001 (one-way ANOVA with Dunnett multiple comparisons test compared with the non-treated 0 control group). B Live/dead analysis of MB spheres treated with different concentrations of mibefradil. Generated spheres were grown for 5 days and treated for 3 days. Single-cell suspension from dissociated spheres was stained with calcein-AM/propidium iodide and imaged using IN-Cell 2200 analyzer (20 × magnification). Left panel: exemplary fluorescence images. Right panel: quantitative analysis of acquired images using IN Carta image analysis software. The scale bar is 60 μm. Results for each concentration were standardised to the non-treated control and expressed as fold change. Data expressed as mean ± standard deviation from three independent assays with three replicates each. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-way ANOVA with Dunnett multiple comparisons test compared with the non-treated control group)