Fig 2: The endosomal localisation of Htt and septins are perturbed in HeLa cells expressing GFP-SNX21 binding mutants. (A,B) HeLa cells were transiently transfected with constructs encoding GFP-SNX21 or respective GFP-SNX21 mutants that do not bind to Htt or septins. Cells were fixed and immunostained for endogenous Htt, septin 7 or septin 9. (C-E) Pearson's correlation co-efficients between GFP-SNX21 and endogenous proteins were quantified from digital confocal images using Volocity. GFP-SNX21 (D48K, D49K) demonstrated a quantifiable loss in Pearson's correlation with Htt when compared with the wild-type protein, as was observed for GFP-SNX21 (L363A) with respect to septin 7 and septin 9. Graphs represent the mean of three biological replicates, with =7 cells quantified from each. Error bars indicate s.e.m; scale bars: 20 µm. An unpaired t-test was used to compare the significance of change with respect to the amount of Htt, septin 7 or septin 9 precipitated with wild-type and mutant SNX21.

Fig 3: MSD signal ratio for mHTT by total HTT, corresponding to the average polyQ length, correlates with average CAG repeat length. (a) Striatal homogenates from 14 homozygous HdhQ140 KI mice of different ages were analyzed by MSD assay for average polyQ length quantification (MSD signal ratio MW1/MAB5492 corresponding to mHTT/Total HTT). Results were plotted as a function of average CAG repeat length determined by PCR method in DNA extracted from the contralateral striatum of each animal (see Methods, Quantification of average CAG repeat length). It is unclear why there is more variability (larger SDs) in raw MSD signals for samples between ~108 and 124 CAG repeats than for other samples. All samples were processed at the same time and in the same manner, so it is likely that variation may be from pipetting. (b) Homogenates prepared from postmortem cortex of HD patients were analyzed by MSD assay for average polyQ length quantification (MSD signal ratio for mHTT by total HTT). Results were plotted as a function of average CAG repeat length determined by PCR method from the same sample lysates (see Methods, Quantification of average CAG repeat length). Light blue sample was below the level of detection (background + 3 SD) for total HTT assay and was not used for correlation. Mean values ± propagated SD (1 s) of duplicates of a single experiment are shown. Please note that the MSD signal is normalized using the following MSD signal ratio (MSD of mHTT/MSD of Total HTT; plotted on y-axis in the figure). The MSD signal for total HTT is solely dependent on protein concentration and does not depend on polyQ length.

Fig 4: PolyQ length affects GST-FLAG-HTTexon1 quantification by MSD assay using polyQ targeting detection Ab. (a) Diagram shows antibody epitopes in human HTT protein (NCBI reference sequence: NP_002102.4). Calibration curve performance for GST-FLAG-HTTexon1 protein using MW1 (b) and MAB5492 (c) detection Abs. Curves were fitted with a four-parameter logistic regression model with 1/Y2 weighting. Mean values ± SD (1 s) of duplicates of a single experiment are shown. (d) Plot of ratio of the slopes determined from standard curves in the linear dynamic range for mHTT assay by total HTT assay as a function of polyQ length exhibits a strong correlation. Mean values ± propagated SD (1s) of duplicates of a single experiment are shown. (e) Using the polyQ length-dependent correlations shown in (d), MSD signal fold increase as a function of polyQ length at constant amount of mHTT protein was extrapolated for mHTT assay. mHTT signal predicted for GST-FLAG-HTTexon1 proteins from Q38 to Q62 was normalized by the MSD signal for GST-FLAG-HTTexon1-Q38. PolyQ lengths ranging from Q38 to Q62 correspond to the polyQ length range seen in adult HD patients. GST: glutathione S-transferase; N17: HTT first 17 aa; PRD: proline-rich domain.

Fig 5: HTT expression was inversely related to miR-146a expression in OSCC tissues. (A) The binding site of miR-146a in the HTT gene. (B) Pearson analysis showing the correlation between miR-146a and HTT mRNA expression level in OSCC tissues (n = 14; r = -0.7022, P < 0.01). HTT expression pattern in (C) miR-146a transfected, and (D) miR-146a knockdowned OSCC cells. Data are presented as mean ± SD form three independent experiments in triplicate. Significant effect of treatment, *P < 0.05, **P < 0.01, and ***P < 0.001. NC, negative control. Scale bar, 100 µm.