Fig. 4.

The role of APPL1 in aggresome formation and clearance. (A) HeLa cells seeded on coverslips were transiently transfected with GFP-APPL1; 24 h later they were treated with 10 μΜ MG132 for additional 6 or 20 h, then fixed and stained for GFP and endogenous ubiquitin. Insets show a magnification of aggregates of APPL1 from cells treated with MG132 for 20 h. Bar, 20 μm. (B) HeLa cells seeded on coverslips were transfected with 10 nM siRNA (either control or targeting APPL1). After 24 h cells were treated with 5 μM MG132 for 18 h, fixed and co-stained for ubiquitin and APPL1. Bar, 20 μm. (C) HeLa cells seeded on coverslips were transfected with two independent control siRNAs and two siRNA duplexes against APPL1. After 24 h cells were treated with 5 μM MG132 for 18 h, washed and allowed to grow in normal medium for additional 24 h. Cells were fixed and co-stained for ubiquitin, APPL1 and DAPI. Cells containing clusters of ubiquitin were grouped into four types based on their size (representative images are shown in upper panel; scale bar 20 μm). The cells were counted and expressed as a percentage of scored cells (total number of cells n = 566 for control siRNAs and n = 713 for APPL1 siRNAs). The graph shows averaged results obtained from two independent siRNA sequences with standard deviation. *, p < 0.05; ns, not significant compared with control siRNA (Mann–Whitney test). Right panels show the efficiency of APPL1 knockdown in comparison to α-tubulin and β-actin levels in cells treated with control (ctrl) and APPL1 siRNA duplexes. The cells used for Western blot were grown and treated in parallel to the cells used for microscopical analysis and they were either lysed or fixed at the same time.