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. 2020 Oct 16;10:17589. doi: 10.1038/s41598-020-74613-9

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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 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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Caspase 3 inhibition protects spine density and architecture in oligomycin A treated neurons. (a) Attenuated oligomycin A-induced dendritic protrusion reduction in 1 µM Q-VD-OPh treated hippocampal neurons. (a1) Dendritic protrusion of vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated hippocampal neurons. (a2–a5) Density of multiple types of spine in vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated hippocampal neurons (a2) Mushroom spine, (a3) Stubby spine, (a4) Filopodia, (a5) Thin spine. Two-way ANOVA followed by Bonferroni post hoc analysis; *P < 0.05, **P < 0.01 and ***P < 0.001. n = 10–15 dendritic segments. (a6) Representative images of dendritic protrusions (green). Scale bar 5 µm. (b1) Cleaved Caspase 3 expression levels in dendritic protrusions of vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated hippocampal neurons. Two-way ANOVA followed by Bonferroni post hoc analysis; *P < 0.05. n = 15–30 dendritic protrusions. (b2) Representative images of Cleaved Caspase 3 (red), dendritic protrusions (green) and MAP2 (blue). Scale bar 5 µm. (c) Mitochondrial membrane potential of vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated hippocampal neuron dendritic (c1) and somatic (c2) mitochondria in the indicated treated hippocampal neurons. Two-way ANOVA followed by Bonferroni post hoc analysis; *P < 0.05, NS not significant, n = 35–40 dendritic segments, respectively. (c3) Representative images of TMRM. Images were recorded at time 0, 5, 10, 15, 20, 25, 30 min. Scale bar 50 µm. (d) Hippocampal neuronal ATP production under vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated. Two-way ANOVA followed by Bonferroni post hoc analysis; NS, not significant, *P < 0.05. n = 6 samples. (e1) Hippocampal dendritic mitochondrial ROS levels of vehicle, 1 µM Q-VD-OPh and/or 1 µM oligomycin A treated neuron cultures. Two-way ANOVA followed by Bonferroni post hoc analysis; NS, not significant, ***P < 0.001, n = 20–30 dendritic segments. (e2) Representative images of Mitosox Red staining. Mitochondria are labeled with Mitotracker green. Hoechst was applied for nucleus staining. Scale bar 20 µm.