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. 2018 Jul 2;8:9930. doi: 10.1038/s41598-018-27842-y

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© The Author(s) 2018

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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Aβ_25–35-induced cathepsin B acts upstream of DAPK1. (A) The effects of cathepsin B inhibitor (5 μM) treatment on the expression of DAPK1 and p-MLC in LPS-primed, Aβ_25–35-stimulated Bv2 cells were analyzed by western blotting analysis (see original blots in Supplementary Fig. S5A). (B) Cells were transiently transfected with DAPK1 overexpression plasmids (cDAPK1) or empty plasmids (Trans control) using Lipofectamine 2000. The effect of cathepsin B inhibitor (5 μM) treatment on Aβ_25–35-induced IL-1β secretion in DAPK1 overexpression cells were analyzed by ELISA. (C–D) LPS-primed Bv2 cells were treated with or without DAPK1 inhibitor (10 μM), followed by Aβ_25–35 exposure for 24 h. (C) The expression of cathepsin B in the cytoplasm was determined by western blotting analysis (see original blots in Supplementary Fig. S5B). (D) Fluorescent images of acridine orange and a cathepsin B substrate were exemplified (12 images per condition). Scale bar = 20 μm. Data are shown as mean ± SEM for at least three independent experiments. ^*P < 0.05 and ^***P < 0.001; ^###P < 0.001. AO: acridine orange; Cat B: cathepsin B substrate; Cat B inh: cathepsin B inhibitor; cDAPK1: constructively activated DAPK1 (DAPK1^ΔCaM mutant); DI: DAPK1 inhibitor; Trans con: transfection control.