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. 2016 Jan 4;6:18512. doi: 10.1038/srep18512

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Copyright © 2016, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) miRNA-based plasmids effectively knockdown PARP6 protein levels. HEK 293T cells were co-transfected with HA-PARP6 and GFP-expressing miRNA-based knockdown plasmids targeting rat PARP6 (miR-PARP6-1, 2, or 3) or LacZ (miR-LacZ). Proteins were resolved by SDS/PAGE and detected by Western blot with anti-HA and anti-GFP antibodies. (b) Knockdown of PARP6 using miRNA-based plasmids decreased dendritic complexity. E18 primary rat hippocampal neurons were transfected with miRNA-based knockdown plasmids on DIV7 and fixed on DIV12. Shown are representative binary images generated from fluorescent images. Scale bar, 20 μm. (c) Quantification of results in (b) using Sholl analysis. Error bars represent SEM. *p < 0.05, **p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-LacZ. (d) PARP6^Res is resistant to knockdown by miR-PARP6-1. HEK 293T cells were co-transfected with HA-PARP6^WT or HA-PARP6^Res. Proteins were resolved by SDS/PAGE and detected by Western blot with anti-HA and anti-GFP antibodies. (e) PARP6^Res rescues miR-PARP6-1-mediated decrease in dendritic complexity. E18 primary rat hippocampal neurons were transfected either with miR-LacZ, miR-PARP6-1, or miR-PARP6-1 and HA-PARP6^Res on DIV7 and fixed on DIV12. Shown are representative binary images generated from fluorescent images. Scale bar, 20 μm. (f) Quantification of results in e using Sholl analysis. Error bars represent SEM. *p < 0.05, **p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-LacZ only. ^#p < 0.05, ^##p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to PARP6^Res + miR-PARP6-1. (g) PARP6^Res restores miR-PARP6-1-mediated decrease in dendritic branching. Quantification of total number of dendrite branch points from neurons described in error bars represent SEM. **p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-LacZ; ^##p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-PARP6-1. (h) PARP6^Res restores miR-PARP6-1-mediated decrease in dendritic length. Quantification of total dendrite length from neurons described in (e). Error bars represent SEM. **p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-LacZ; ^##p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to miR-PARP6-1.