Fig 1: p53 protein levels in iPSC and NSC states are dependent on mutant HTT levels. (A,B) The decreased p53 level is maintained in HD YAC128 NSCs after differentiation of iPSCs, as assessed by western blotting (N = 4 for WT and N = 5 for YAC128). The data was normalized to total protein visualized on blots using Bio-rads stain-free technology. Data from YAC128 iPS cells (X) was adapted from our previous work (Szlachcic et al., 2015) (N = 6 for WT, N = 5 for YAC128). shHTT iPSC (C) and NSC (D) lines show reversal of the p53 phenotype, whereas shCAG reagents further decrease the p53 expression level. N = 4 for each reagent in both iPSC and NSC. (E) Pearson correlation reveals an inverse correlation of mutant HTT and p53 expression levels (N = 11 for HTT reagents and N = 9 for control shCTRL reagent). In NSCs, the correlation is not significant **p < 0.01, ***p < 0.001. In Panel (A) and (C, iPSCs) blots were cropped; full-length blots are presented in Figure S8.
Fig 2: INT41 reduced Htt aggregate formation in R6/2 animal model: aggregates in coronal sections of female mice were measured in 4 fields, binned by size, and analyzed for the frequency of sized aggregates as indicated. Analysis of variance on 4113 data points for 0-1 µm size category was 0.085 comparing striatal fields, as described in Section 2, from 5 GFP and 6 INT41 mice.
Fig 3: In the presence of SIP, the levels of N-terminal HTT decrease and mutant HTT aggregates decrease. a SIP decreases the expression of proteins encoded by exon 1 of wt HTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP. b SIP decreases the aggregation of proteins encoded by exon 1 mHTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP. The left parts of panels a and b were imaged under a fluorescent microscope. Images in the right parts of panels a and b were taken under transmitted light. Scale bar = 1 µM. The size and number of mHTT aggregates (e1HTT-72Q) are presented in panels b’ and b”, respectively. Red color signals of e1HTT-72Q were measured as the number of pixels per aggregate b’ and show the average aggregate size measured for each of the analyzed images. c, d Western blot (WB) analysis of HEK293T cells that co-expressed e1HTT-25Q c or e1HTT-72Q d and wt SIP or control pEntry plasmids. c, d Graphs show densitometric analysis of western blotting bands for the indicated proteins using GAPDH abundance for normalization and presented as fold changes. M, protein marker in kilodaltons (kDa). The results are expressed as mean ± SEM. *p < 0.05, ***p < 0.001. The results were obtained from at least three independent HEK293T culture preparations
Fig 4: Htt levels in subcellular fractions. Whole brain lysates from 6-month-old Htt+/+ (+/+), Htt?Q/?Q (?Q/?Q), Htt?QP/?QP (?QP/?QP), and Htt?N17/?N17 (?N17/?N17) mice were separated into nuclear, cytosolic, and microsomal fractions. No significant differences in Htt protein levels were observed between the homozygous mutants and wild type controls in any of these fractions. For quantification, Htt (MAB2166) levels were first normalized to mTOR levels in the microsomal and cytosolic fractions, and to Lamin B1 levels in the nuclear fractions. The normalized Htt levels in the Htt?Q/?Q, Htt?QP/?QP, and Htt?N17/?N17 samples were then compared to normalized wild type Htt levels. Nuclear fraction: Htt?Q/?Q: 1.062±0.459, Htt?QP/?QP: 0.669±0.198, Htt?N17/?N17: 0.616±0.208; p = 0.6421. Cytosolic fraction: Htt?Q/?Q: 0.999±0.085, Htt?QP/?QP: 0.832±0.090, Htt?N17/?N17: 0.730±0.078; p = 0.0781. Microsomal fraction: Htt?Q/?Q: 0.861±0.166, Htt?QP/?QP: 0.505±0.148, Htt?N17/?N17: 0.851±0.150; p = 0.1591. (mean±SEM, 1-way ANOVA, n = 3-4/genotype).
Fig 5: Effects of mutant HTT knockdown on MAPK and Wnt pathways in mouse iPSCs. (A,B) Western blot analysis of shHTT2 effects on the Wnt pathway revealed a significant increase in total ß-catenin levels but not phospho-ß-catenin (S33/37) levels. N = 4 lines for each reagent; (C,D) Isogenic lines (N = 2 for each reagent) originating from separate parental lines (1 and 2) show different responses to bFGF-induced activation of ERK1/2 phosphorylation (Thr202/Tyr204) after 30 min of stimulation. *p < 0.05, ***p < 0.001. In Panel (A) blots were cropped; full-length blots are presented in Figure S8.
Supplier Page from MilliporeSigma for Anti-Huntingtin (N-terminal) antibody produced in rabbit