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. 2021 Oct 4;58(12):6647–6669. doi: 10.1007/s12035-021-02567-8

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© The Author(s) 2021

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 — Contribution of presenilins to the production of hexameric Aβ assemblies. a SH-SY5Y knockdown (KD) cell lines were generated using CRISPR-Cas9, with guide RNA vectors targeting either human PSEN1 (PS1-KD) or PSEN2 (PS2-KD) genes. Control cells were transfected with respective scrambled sequences. Left, a representative Western blot; middle and right, quantitative decrease in PS1 and PS2 protein levels in KD compared to S cells (for all conditions, see Supplementary Fig.S2). N = 3. One-sample t test with hypothetical value set as 100: *p < 0.05, ***p < 0.001 (S vs KD, in PS1: p = 0.03; in PS2: p = 0.0001). WT wild-type, S scrambled. b, c Initial cleavage ability was monitored by a reporter gene assay. The release of APP intracellular domain (AICD) from a tagged C99-GVP substrate was measured by the Gal4-Firefly reporter gene. Results are represented as Firefly/Renilla luciferases ratios, with Renilla serving as a transfection-efficiency control. The profile of Aβ production was assessed after transfection with either an empty plasmid (EP) or C99, using Western blotting and ECLIA immunoassay, in PS1-KD vs PS1-S (in b) and in PS2-KD vs PS2-S (in c). Media samples were lyophilized prior to analysis. Dashed lines indicate that proteins were run on the same gel, but lanes are not contiguous. Luciferase assays (initial cleavage of C99): N = 4 each. One-way ANOVA with Tukey's multiple comparison test: ns = non-significant (S vs KD, in PS1 control: p > 0.99; in PS1 C99-GVP: p = 0.10; in PS2 control: p = 0.99; in PS2 C99-GVP: p = 0.99). Western blots quantitative analyses (hexameric Aβ, in cell lysates and released, relative to C99 and expressed as a % to S): N = 3 each. One-sample t test with hypothetical value set as 1: ns non-significant, **p < 0.01 (S vs KD, in PS1 cell lysates: p = 0.16; in PS1 media: p = 0.97; in PS2 cell lysates: p = 0.99; in PS2 media: p = 0.01). ECLIA assays (monomeric Aβ released): N = 5 each. Mann–Whitney test: ns non-significant, *p < 0.05 (S vs KD, in PS1 Aβ₄₀: p > 0.99; in PS1 Aβ₄₂: p > 0.99; in PS2 Aβ₄₀: p = 0.04; in PS2 Aβ₄₂: p = 0.12)