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. 2020 Apr 23;26(5):1606–1618. doi: 10.1038/s41380-020-0728-2

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© The Author(s) 2020

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. 3 — a RT-qPCR analysis of COSMOC and MOCOS expression during neuronal differentiation of SH-SY5Y cells for 9 days in vitro (DIV). Gene expression was normalized to time-point 0 (n = 3). b Neuronal differentiation was confirmed by RT-qPCR analysis showing decreased expression of the cell division marker CDK1 and increased expression of the neuronal marker MAPT. Data were normalized to time-point 0 (n = 3). c Immunocytochemistry with the neuronal differentiation markers βIII tubulin and MAP2 on undifferentiatied and differentiated SH-SY5Y cells at 9 days. d RT-qPCR analysis showing effective depletion of COSMOC in SH-SY5Y cells during the first 5 days of neuronal differentiation. SH-SY5Y cells treated with si-COSMOC were compared with cells incubated with scrambled siRNA and data were normalized to time-point 0 (n = 3). e, f RT-qPCR confirmation of effective neuronal differentiation of SH-SY5Y depleted in COSMOC expression during the first 5 DIV. The expected inverse correlation between CDK1 expression (e) and MAPT expression (f) was observed in SH-SY5Y treated with si-COSMOC and those treated with scrambled siRNA. Data were normalized to time-point 0 (n = 3). g Immunocytochemistry showing βIII tubulin and MAP2 staining of SH-SY5Y cells treated with siCOSMOC, siMOCOS or control siRNA at 5 DIV. h Analysis of cell death by nuclear staining with Hoechst. Example of a nucleus counted as apoptotic after staining with Hoechst (leftmost) and compared with two adjacent normal nuclei. i Quantification of apoptotic nuclei at 5 DIV in SH-SY5Y knockdown for COSMOC, MOCOS or control siRNA (n = 4; Mann–Whitney test: *p < 0.05). Representative Western blot (j) and quantification (k) of cleaved products of caspase-3 (17 and 19 KDa) at 5 DIV in SH-SY5Y cells depleted in COSMOC, MOCOS or control siRNA. GAPDH was used as control. (n = 4; Mann–Whitney test: *p < 0.05). Data are represented as mean ± SEM. For all figures, n stands for number of biological replicates of SH-SY5Y cells.