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. 2020 Oct 17;11(10):874. doi: 10.1038/s41419-020-03085-6

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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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Fig. 3 — a, b Ubiquitin and p62 levels in differentiated SH-SY5Y cells treated with amitriptyline and MG132 were analyzed by western blotting. SH-SY5Y cells were treated with MG132 in a two-step process (pro-incubated with a low dose of 0.1 μM for 16 h and changed to fresh normal media; after 8 h, incubated with a high dose of 1 μM for additional 16 h). Amitriptyline was applied 1 h prior to MG132 high-dose treatment. Quantification of ubiquitin and p62 in the SDS-fractions in a is shown in the right panel (n = 3–4). The expression and localization of ubiquitin (magenta) and p62 (green) in SH-SY5Y cells treated with MG132 and amitriptyline were analyzed by immunofluorescence (b). Scale bar corresponds to 50 μm. Quantification of ubiquitin and p62 immunofluorescence intensity in b is shown in the right panel. Percentage of aggresome (spots larger than 2 μm in diameter) in total cell is shown in the right panel (n = 60–100) c SH-SY5Y cells were treated with 1 μM MG132 in a two-step process and 20 μM amitriptyline as described in (a) and incubated with fresh media for an additional 24 h after MG132 treatment. Amitriptyline groups were treated amitriptyline in fresh media. Ubiquitin and p62 protein levels were analyzed by western blot. Protein levels in the SDS-fraction were quantified by densitometric analysis (n = 4–5). d The ubiquitin and p62 aggregates in MG132 and amitriptyline co-treated cells after the recovery period was evaluated by immunofluorescence. Scale bar corresponds to 10 μm. (n = 25–30) Quantification of ubiquitin and p62 immunofluorescence intensity in (d) is shown in the right panel. e, f SH-SY5Y cells were pretreated with amitriptyline for 1 h and treated MG132 in a two-step process, followed by a recovery period for 48 h. Cell death was assessed by LDH assay (e) and MTT assay (f) (mean ± S.E.M.; n = 7–8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AMI amitriptyline, CON control, NP-sol NP-soluble fraction, SDS-sol SDS-soluble fraction, Ub ubiquitin.