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Fig. 6. (a) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 48 hours were subsequently transfected with EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (Q74) for 24 hours. Quantification of Q74 expressing cells with aggregates is shown in the graph. *P<0.01. (b) HeLa cells stably expressing GFP-LC3 were transfected with control or clathrin siRNA for 72 hours, numbers of LC3-positive vesicles were counted (>20 vesicles per cell); ***P<0.0001. (c) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 72 hours were subjected to western blot analysis using anti-LC3 and anti-actin antibodies. Under exposure conditions that allowed endogenous LC3-II quantification in these cells, the LC3-I signal was frequently too low to be detected. Quantification of band intensities from three independent experiments is shown. *P<0.05. (d) HeLa cells transfected with control siRNA (Cont) or siRNA targeting clathrin heavy chain (Cla) for 48 hours were subsequently transfected with HA-Atg12 and Atg5 for a further 24 hours. Western blot analysis was performed with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (e) HeLa cells stably expressing GFP-LC3 were left untreated (Cont) or treated with 5 mM methyl-β-cyclodextrin (β-CD), in the presence (+) or absence (–) of bafilomycin A1 (BafA1) for 6 hours were subjected to western blot analysis using anti-GFP (to detect LC3) and anti-actin antibodies. (f) Lysates from HeLa cells transfected with empty vector (Cont), DN-Rab5 or CA-Rab5 were blotted for phosphorylated (S6-P) and total (S6-T) ribosomal protein S6. Lysate from cells treated with rapamycin (Rap) was used as a positive control. (g) HeLa cells transfected with control vector (Cont) or wild-type Rheb together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (h) Schematic hypothetical representation of how autophagy is regulated by Rab5. Our data suggest that the Atg5 structures are probably precursors of the pre-autophagosomal structures. The accumulation of Atg5 structures that were observed by us following inhibition of Rab5 or Vps34 might be owing to a block in the progression from early Atg5-positive autophagosomal structures to the formation of autophagic vacuoles.
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