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

Figure 7.

Figure 7

Hipk3 regulates mHtt levels via DAXX. (A) Left, RT-qPCR and western blots confirming the knockdown of DAXX in HD patient fibroblasts (Q68) by its siRNA. Right, mHTT levels in HD patient fibroblasts (Q68) measured by HTRF using the antibody pair 2B7/MW1, after transfection of the indicated siRNAs. Transfection of the DAXX siRNA blocked the effect of knocking down HIPK3. (B) mHTT levels in HD patient (Q47) primary fibroblasts (left) or HD patient iPSC-derived neurons (right) measured by HTRF using the antibody pair 2B7/MW1, after transfection of the indicated siRNAs. Transfection of the DAXX siRNA blocked the effect of knocking down HIPK3. (C) Representative images and quantification of endogenous LC3II puncta in patient iPSC-derived neurons (Q47) transfected with the indicated siRNAs. Scale bar, 20 μm. The quantification of the puncta was performed blindly, the boxed regions have been magnified and exhibited at the lower right corner for better view of the puncta, but the counting was not limited to the boxed regions. The statistical analysis was performed by two-way unpaired t tests. (D) Representative western blots and quantification of p62 and LC3 blots of patient iPSC-derived neurons (Q47) transfected with the indicated siRNAs. (E) Representative western blots and quantification of DAXX in patient iPSC-derived neurons (Q47) transfected with the indicated siRNAs. (F) A model illustrating the positive feedback loop mediating MAPK11 and HIPK3's effect on mHTT levels. For all bar plots, data represent mean and SEM. The statistical analysis was performed by two-way unpaired t tests for A to D, and by one-way ANOVA followed by Dunnett's post hoc tests for E and F. *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05.