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. 2018 Jul 17;9:2773. doi: 10.1038/s41467-018-05124-5

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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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Fig. 1 — Dendrite development is impaired in neurons depleted of APC2. a Representative images of dendritic morphology of neurons transfected with MARCKS-BFP for cell outline and either control or APC2 shRNA with MARCKS-BFP. b mRNA levels in hippocampal neurons after 3 days of either control or APC2 shRNA expression. Data normalized to GAPDH (n = 3, p = 0.008, One sample t-test). c Dendrite thickness was measured within 5 µm of the cell body (n = 188 dendrites, p = 0.963 DIV11, p = 0.206 DIV18, N = 2, Mann–Whitney U test). d Number of primary dendrites was quantified per cell body (n = 39 neurons control shRNA DIV11, n = 41 neurons APC2 shRNA DIV11, p = 0.009, n = 40 neurons DIV18, p = 0.020, N = 2, Mann–Whitney U test). e Sholl analysis of dendritic branching at DIV11 (n = 39 neurons control shRNA, n = 41 neurons APC2 shRNA, N = 2, Two-Way ANOVA, Tukey post-hoc). f Sholl analysis of dendritic branching at DIV18 (n = 40 neurons, N = 2, Two-Way ANOVA, Tukey post-hoc). g Representative images at DIV18 of dendritic spines in control or APC2 shRNA with MARCKS-BFP transfected neurons. Homer and Bassoon levels in dendritic spines between control and APC2 depleted neurons did not differ. (n = 42 spines control shRNA, n = 37 spines APC2 shRNA; Homer: control shRNA 6797 ± 470 fluor. units vs. APC2 shRNA 8360 ± 783 fluor. units, p = 0.125, Mann–Whitney U test; Bassoon: control shRNA 16670 ± 923 fluor. units vs. APC2 shRNA 16441 ± 1348 fluor. units, p = 0.589, N = 2, Mann–Whitney U test). h Total number of spines positive for Homer and Bassoon quantified per 50 μm length of dendrite at DIV18. (n = 50 dendrites control shRNA, n = 42 dendrites APC2 shRNA, p ≤ 0.001, N = 2, Independent samples t-test). i Number of spines positive for Homer and Bassoon was classified and quantified per 50 μm length of dendrite at DIV18. Mushroom spines are > 2 μm long with mushroom shape (p ≤ 0.001), filopodia are > 1 μm with no thickening (p = 0.001) and stubby spines are ≤ 1 μm (p ≤ 0.001). Other protrusions (p = 0.091) were those that were negative for Homer and/or Bassoon staining (n = 50 dendrites control shRNA, n = 42 dendrites APC2 shRNA, N = 2, Mann–Whitney U test). Graphs represent mean ± SEM. * p ≤ 0.05. Scale bars are 10 μm in a and 5 μm in g