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. 2020 Jul 16;10:11827. doi: 10.1038/s41598-020-68794-6

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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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Candidate targets validation. (a) Schematic representation of the luciferase reporter assay used for validation of FUS^P525L regulation of protein levels via 3′UTR binding. (b) Luciferase assay on HeLa cells expressing RFP-FUS-WT and RFP-FUS-P525L and transfected with a luciferase construct containing the 3′UTR of the indicated candidates. Histogram bars represent the average of 3 experiments and error bars indicate the standard deviation (Student’s t test; paired; two tails; *p < 0.05; ***p < 0.001). Values have been normalized to the average of FUS-WT set as 1. (c) Western blot analysis of selected targets (ASAP1; VCP) and controls (neuronal tubulin beta 3, TUBB3; GAPDH) in hiPSC-derived motoneurons. (d) Quantification of Western blot signals of the indicated candidates. TUBB3 signal was used for normalization. Histogram bars represent the average of 3 independent differentiation experiments (shown in Supplementary Fig. S3 online) and error bars indicate the standard deviation (Student’s t test; paired; two tails; *p < 0.05).