Figure 2

Effects of hook2 on aggresomes formation. (A) Vero cells were transfected to express CFTR. Immunofluorescence staining revealed CFTR either diffusely distributed □, aggregated , or accumulated in aggresomes ■. (B-C) Cells were transfected to express ΔF508-CFTR with or without hook2. After 24 h 10 μM lactacystine was added for the indicated times, before cells were stained for CFTR and the fractions of cells exhibiting distinct patterns were counted. Pie charts show an increase in the percentage of cells with aggresomal CFTR in cells that co-express hook2, compared to cells that express ΔF508-CFTR alone. Even though lactacystin treatment increased the incidence of ΔF508-CFTR in aggresomes, hook2 promoted aggresome formation in the absence of lactacystin (0 h). (C) The aggresome-promoting effect of hook2 was even more pronounced when cells in which hook2 was enriched at centrosomes were considered separately from those in which hook2 was distributed diffusely. (D-E) Cells were transfected to express hook2 alone or with ΔF508-CFTR and the fraction of cells with type IV hook2 distribution (See Fig. 1A) was counted. (D) In contrast to the effect of hook2 on CFTR (shown in B and C), the presence of CFTR did not change the percentage of cells with type IV hook2. 6 or 12 hours of lactacystin exposure increased the percentage of cells with hook2 aggresomes, but CFTR had no additional effect. (E) CFTR distribution strongly correlated with that of hook2 since in 100% of cells with aggresomal CFTR hook2 was co-enriched at centrosomes.