Fig 2: Screening for modulators of Htt levels in the striatal cells (STHdh).(A) Representative western-blots in the STHdhQ7/Q111 cells of candidate modifiers of mHtt levels. 104 candidate modifiers were selected based on our previous screening results (doi: 10.1038/nn.3367). The genes that had averaged Z score values larger than 1.2 and were not previously identified as validated hits in the patient fibroblasts were selected for testing in STHdh cells by pooled siRNAs (Dharmacon, custom library). The MW1 detected full length mHtt is quantified and normalized to the tubulin signal using the ImageJ software. The siRNAs that change mHtt levels by more than 30% in the same direction in three biological repeats were selected as hits. The number on top of each bar indicates the biological repeat number of siRNA knock-down. (B) The bar plot of mHtt level changes upon knock-down of the identified modifiers. The numbers on top of each bar represent the number of biological replicates tested.DOI: http://dx.doi.org/10.7554/eLife.05449.004

Fig 3: PolyQ-dependent signal increases with longer glutamine stretches. HEK cells were transiently transfected with full-length HTT expressing different polyQ lengths. The ratio between polyQ-dependent and -independent assay signal was calculated as average of the ratio in three different experiments where the average values are indicated in each bar for ease of reading. Data are presented as mean ± SEM.

Fig 4: Purified HTT protein standards. Serial dilutions of full-length purified human HTT protein expressing different polyQ were used to generate standard curves with the polyQ-dependent and -independent assay (A, B). Lower limit of quantification (LLoQ) was 3 pm per well for both assays and it is indicated by a dashed line on the graphs. polyQ-dependent increase in signal intensity is displayed as the ratio of the raw AlphaLISA counts in polyQ-dependent/-independent assay at 100 pm in each well (C). Hook points were calculated using serial dilutions of the biotinylated CST5656 in the presence of constant Acceptor beads-conjugated antibody and purified HTT protein with 23 or 78Q (D, E). Best assay conditions are indicated with a dashed line at 1 nm of the biotinylated antibody. Data in A, B, D, and E are presented as mean ± SEM.

Fig 5: Lowering Mapk11 rescues HD-relevant phenotypes in iPSC-derived HD neurons and HD knockin mouse model. (A) Left panels, representative phase-contrast (for cell confluence and morphology) and green fluorescence images (for caspase-3 activity using the caspase-3 detection dye) of the wild-type (WT, Q19) or the Huntington's disease (HD, Q47) iPSC-derived neurons transfected with the indicated siRNAs under BDNF deprived conditions for 48 h. Scale bar, 100 µm; Right panel, quantification of the number of caspase-3 signals (green particle counts × size × mean intensity) per field by IncuCyte software. The numbers on top indicate the number of independently transfected wells. 4 fields of each well were imaged for quantification by IncuCyte inside the cell culture incubator. All the signals were normalized to the average of the HD cells. Bars represent mean and SEM. The statistical analysis was performed by one-way ANOVA and Dunnett's post hoc tests. (B) Left panels, representative images of the Htt aggregates (by the antibody S830) alone or merged with DAPI and Darpp32. The scatter plot, quantification of the total aggregate signals (aggregate counts × size × mean intensity) per cell by particle analysis using ImageJ. Data were from 12 different slices from 3 mice of each genotype. Scale bar, 20 µm. The imaging and the analysis were performed blindly before revealing the genotypes. The statistical analysis was performed by one-way ANOVA and Dunnett's post hoc tests. (C) The total number of active behaviors (activities) in 5 min for each mouse of the indicated genotypes tested in the pen holder with mashed surface. Unpaired two-tailed t test was performed between the HD (HdhQ140/Q140) and WT (HdhQ7/Q7) mice without Mapk11 deletion (Mapk11+/+); one-way ANOVA and Dunnett's post hoc tests were performed within the HD and WT groups. (D) Left, the total distance traveled in 15 min for mice of the indicated genotypes tested in the open-field. Right, the open-field was divided into 4 × 4 virtue grids for analysis, and the number of times that each mouse crossed the borders of the middle 4 blocks was quantified. The statistical analysis was performed and indicated as in (C). (E) Representative footprints of mice of indicated genotypes. (F) The plot of the top principle component (Coupling PC1) of the gait coupling parameters (Supplementary information, Table S1). The statistical analysis was performed and indicated as in (C). (G) Summary of the number of rescued (green) versus exacerbated (orange) parameters by Mapk11 heterozygous or heterozygous knockout. The category was judged by the direction of changes. Heterozygous or homozygous knockout of MAPK11 in HD mice led to predominantly more rescues (green) than exacerbations (orange), suggesting rescue effects. See Supplementary information, Table S2 for the selected parameters. For all plots, error bars represent mean and SEM. The numbers on top of each plot indicate the number of mice. For all the behavioral experiments and aggregation immunofluorescence, mice were tested blindly and their genotypes were revealed after data recording and analysis. Immunofluorescence data were obtained from 11 month old mice and behavioral data were obtained from 7.5 month old mice.