Figure 5.

Suppression of autophagy enhances accumulation of ubiquitinated proteins and aggregates in cancer cells. A: Bax-deficient HCT 116 cells were transfected with negative siRNA (Neg) or siRNA against Atg8/LC3B or Atg6 for 48 hours and then treated with MG132 (0.5 μmol/L) or with control vehicles for 24 hours. Lysates were prepared in radioimmunoprecipitation assay buffer and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot assay was conducted with the anti-ubiquitin (top panels) and anti-β-actin (bottom panels) antibodies. B and C: HCT 116 Bax-deficient cells were transfected with designated siRNA and treated with MG132 (0.5 μmol/L) for 24 hours (B) or as indicated (C). Cells were then fixed with 4% paraformaldehyde and stained with the anti-ubiquitin antibody and the DNA dye Hoechst 33342. Dispersed ubiquitinated proteins could be detected in the nucleus of nontreated cells, but the perinuclear ubiquitinated protein aggregates could be only detected in MG132-treated cells (arrows). The latter (aggresome-positive cells) were quantified and expressed as the percentage of total anti-ubiquitin-positive cells (C). *P < 0.01 and ^$P < 0.05 by Z-test (specific siRNA versus negative siRNA). D: DU145 cells stably expressing GFP-LC3B were treated with bortezomib (10 nmol/L) for 24 hours and then immunostained with an anti-ubiquitin antibody as well as Hoechst 33342 as above. Cells were then subjected to confocal microscopy. a shows the GFP-LC3B puncta, whereas b shows the ubiquitin-positive aggresomes. Merged image in c indicates the colocalization of GFP-LC3B puncta and the aggresomes.