Figure 3.

(See previous page). Protein aggregate clearance by selective autophagy requires MYO1C. (A) HeLa cells stably expressing HTTQ72-GFP were transfected with siRNA targeting MYO1C or treated with 1 μM PCIP, alongside appropriate controls, and labeled for immunofluorescence using antibodies to GFP. Bar = 10 μm. (B) HTTQ72-GFP aggregates were quantified in mock-, MYO1C siRNA-, DMSO-, 1 μM PCIP-, and 100 nM bafilomycin A₁-treated cells. The results are represented as the percentage of HTTQ72-GFP-expressing cells with more than 15 GFP-positive spots per cell. MYO1C knockdown and PCIP treatment lead to a significant increase in the number of cells containing HTTQ72-GFP aggregates. Graphs represent the means ± s.e.m from 3 independent experiments. A total number of >3900 cells was analyzed. (C) HeLa cells stably expressing HTTQ72-GFP were treated with 100 nM bafilomycin A₁ for 16 h prior to processing for immunofluorescence microscopy. Scale bar = 20 μm. (D) HeLa cells stably expressing HTTQ72-GFP were treated with 1 μM PCIP for 16 h prior to processing for immunofluorescence microscopy using antibodies to the indicated proteins. Nuclei in blue were labeled with Hoechst. Scale bar = 20 μm.