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. 2017 Oct 13;27(12):1441–1465. doi: 10.1038/cr.2017.113

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Copyright © 2017 The Author(s) 2017

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HIPK3- and MAPK11- mediated HTT regulation is mHTT-dependent. (A) Representative western blots and quantification of the STHdh^Q7/Q7 (WT) cells showing that expression of the mHTT (Q72) but not the wtHTT (Q25) N-terminal exon1 fragments enables the reduction of endogenous wtHtt levels by knocking down Mapk11. The wtHtt change by the siRNA targeting Mapk11 was calculated within each group of cDNA transfected samples and plotted as the percentage difference between each of the Mapk11 siRNA-transfected well and the average of the scramble siRNA-transfected control within the same group. The statistical analysis was performed by one-way ANOVA and Dunnett's post hoc tests. (B) Similar to (A), but showing the effects of knocking down Hipk3. (C) Representative western blots and quantifications (by ab1) of the HD (STHdh^Q7/Q111) cells showing that specifically knocking down mHtt using the allele-specific siRNA (allele specific mHtt si) abolished the reduction of endogenous Htt levels by knocking down Mapk11 or Hipk3. The statistical analysis was performed by two-tailed unpaired t tests. (D) qPCR measurements of Hipk3 mRNA levels in the HD (Q7/Q111) versus the WT (Q7/Q7) STHdh cells (left), and in the HD (Hdh^Q140/Q7) versus the WT (Hdh^Q7/Q7) mouse striata of animals at indicated ages (middle and right). The levels were normalized to the average of WT samples. The statistical analysis was performed by two-tailed unpaired t tests. (E) qPCR measurements of Hipk3 mRNA levels in the HD (STHdh^Q7/Q111) cells or the WT (STHdh^Q7/Q7) cells transfected with the indicated siRNAs. B01, C01 and Hdh5 are siRNAs targeting Htt (see Supplementary information, Data S1). The levels were normalized to the average of scrambled siRNA-transfected control samples (Scramble), and the statistical analysis was performed by one-way ANOVA followed by post hoc Dunnett's tests. (F) Similar to (E), but in patient iPSC-derived neurons. HTT3 is the siRNA targeting human HTT. (G) Representative western blots and quantifications of phospho-MAPK11/MAPK11 ratio in STHdh^Q7/Q7 WT cells transfected with indicated cDNA and treated with indicated compounds. The p-MAPK11/MAPK11 ratio change compared to the Mock control was quantified to assay the activation of MAPK11. (H) Western blots and quantification of Atf2 and phosphorylated Atf2 (p-Atf2) levels in striata lysates from 6.5-month-old mice with or without Mapk11 knockout in the wild-type background using specific antibodies targeting Atf2 versus phosphorylated Atf2, respectively. Atf2 phosphorylation was significantly decreased in Mapk11 knockout mice, confirming Atf2 as a substrate for Mapk11. Statistical analysis was performed by two-tailed unpaired t tests. (I) Similar to (H), but comparing HD (Hdh^Q7/Q140) versus WT mice (Hdh^Q7/Q7). Atf2 phosphorylation was significantly increased in HD mice, suggesting that Mapk11 activity may be increased. For all plots, error bars represent mean and SEM. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns, P > 0.05.