Fig 7. Truncated HTT lacking the N-terminal region is able to rescue neurite defects of PC12 cells.

(A) Western blotting showing that HTT shRNA depleted Htt expression in PC12 cells, which were also transfected with RFP-tHTT. (B) Immunofluorescent staining of transfected PC12 cells showing RFP-tHTT can reduce the neurite extension defect in cells (red cells with arrows) that also express shRNA-GFP (green) to suppress endogenous Htt, whereas cells with shRNA-GFP alone (green cells in left panel) show defective neurite extension. The cells were treated with NGF (50 ng/ml) for 48 h to induce neurite extension. (C) Transfection of nHTT was unable to prevent Htt loss-mediated neuritic defect. Arrows indicate PC12 cell that expressed nHTT and also shRNA-GFP. Arrowheads indicate PC12 cells with long neurites that only expressed nHTT but not shRNA-GFP. In (B) and (C), scale bars: 10 μm. (D) Quantitation of the number of PC12 cells with neurites longer than two cell bodies. The data are presented as mean±SE (n = 500 cells per group). *** P<0.001. (E). Expression of the truncated HTT (tHTT) or mutant HTT (dHTT) that lacks the dynein-binding region in HEK293 cells. HTT is tagged with Flag and linked to RFP via P2A, a self-cleaving peptide. Western blotting with anti-Flag antibody reveals the expression of tHTT and dHTT. (F). Quantitation of the number of PC12 cells with neurites longer than two cell bodies after inhibiting Htt expression by shRNA and transfection with tHTT or dHTT. The data are presented as mean±SE (n = 500 cells per group). *** P<0.001.