Figure 3.

Rab1 is associated with autophagosomes and its activity is required for their formation. (A) HeLa cells were co-transfected with RFP-LC3 and the indicated CFP-Rab1B constructs, then treated with rapamycin for 2 h to induce autophagy. Magnified images of boxed areas are shown in the upper right corners. Arrows indicate further instances of CFP-Rab1B(Q67L) colocalization with autophagosomes. Size bars, 5 µm. (B) Quantification of the colocalization of CFP-Rab1B constructs with autophagosomes (APs) (RFP-LC3⁺ round vacuoles with clearly visible hollow centers). Asterisks indicate a significant difference, p < 0.05. (C) Cells were transfected as in (A) and either left untreated (−Rap) or treated with rapamycin for 2 h (+Rap). The average number of autophagosomes (APs) per transfected cell was quantified. (D) HeLa cells treated with the indicated siRNAs were transfected with GFP-LC3 and treated with (+) or without (−) rapamycin for 2 h. The number of autophagosomes (APs) per cell was quantified. In a parallel experiment, cells were treated with the autophagy inhibitor WTM during rapamycin treatment. Asterisks indicate a significant difference from control siRNA levels (+Rap), p < 0.05. (E) HeLa cells treated with the indicated siRNAs were incubated in the absence (−) or presence of rapamycin (+Rap), in the presence of bafilomycin A1 (BafA1) alone or rapamycin and bafilomycin A1 together (+Rap+BafA1) for 2 h. Then cells were lysed, harvested and analyzed by western blot. Endogenous LC3-I and LC3-II were detected by a rabbit polyclonal antibody. β-tubulin serves as a loading control. Densitometry was performed using ImageJ software and the ratio of LC3-II to β-tubulin density is plotted below.