Fig 1: PolyQ-huntingtin inhibits ErbB2/HER2 endocytosis through a dynamin dependent mechanismA,B. Human SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100) and treated with Geldanamycin (GA) as indicated. n.t.: not transfected. (A) Cells are stained for HER2 and huntingtin (Htt). HER2 intensity at the membrane is quantified before and after treatment (at least n = 24 cells analysed per condition, three independent experiments). Htt Q23 DMSO vs. Htt Q100 DMSO: *p-value = 0.0204; n.t. GA vs. Htt Q100 GA: ***p-value < 0.0001; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0006. (B) Cells are fixed, immunostained with the antibody recognizing the extracellular part of HER2 and analysed by flow cytometry. A representative flow cytometry profile is shown (left). The graphs in B, C and D represent mean HER2-APC fluorescence level (surface HER2; three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 DMSO versus Htt Q100 DMSO: *p-value = 0.0141; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0009.C. SKBr3 cells are transfected with a construct expressing a GTPase-defective mutant K44A dynamin (Dyn2 K44A), treated with Geldanamycin as indicated and analysed by flow cytometry. (Three independent experiments, 10,000 cells analysed per condition and experiment). DMSO versus GA, ***p-value < 0.0001; DMSO versus Dyn2 K44A DMSO, p-value > 0.9999; GA versus Dyn2 K44A GA, ***p-value < 0.0001; Dyn2 K44A DMSO versus Dyn2 K44A GA, ***p-value = 0.0002. n.s., not significant.D. SKBr3 cells are transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 and a construct encoding wild-type dynamin (Dyn2 WT) and analysed by flow cytometry (three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 versus Htt Q100, *p-value = 0.0491; Htt Q100 versus Htt Q100 + Dyn2 WT, **p-value = 0.0016; Htt Q23 + Dyn2 WT versus Htt Q100 + Dyn2 WT, *p-value = 0.0136.E. Huntingtin and dynamin interact in a polyQ-dependent manner. Huntingtin immunoprecipitation experiments were performed on cellular extracts from MMTV-PyVT/HdhQ7/Q7 (HdhQ7/Q7) and MMTV-PyVT/HdhQ111/Q111 (HdhQ111/Q111) tumours. Immunoprecipitation with mouse IgG (IgG m) is used as a control.F. SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23) and polyQ-huntingtin (pARIS-mCherry-httQ100) and stained for huntingtin (Htt) and dynamin.