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Figure 3


Fig. 3. Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. (a) HeLa cells transfected with control or Atg7 siRNA for 48 hours were subjected to a second round of transfection with GFP-Atg5 together with the siRNA for another 24 hours. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). (b) HeLa cells transfected with control vector or DN-Rab5 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 ratio of Atg5-Atg12 versus free Atg12 is shown in the graph. Data are from four independent experiments; ***P<0.0001. (c) Atg5-Atg12:Atg12 ratio obtained from experiments similar to those shown in b, but performed in the presence or absence (control) of 3-methyladenine (3-MA; four independent experiments; left), or with control siRNA or siRNA targeting Vps34 (three independent experiments; right); ***P<0.0001, **P<0.001. (d) HeLa cells transfected simultaneously with siRNA targeting Rab5a, Rab5b and Rab5c (Rab5; left), Atg7 (right) or control siRNA for 48 hours were subjected to a second round of transfection with Atg5 and HA-Atg12 together with the Rab5 or Agt7 siRNA for another 24 hours. Western blot analysis was performed using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. (e) Immunostaining of HeLa cells transfected with either control (in the prescence or absence of 3-MA) or DN-Rab5 and HA-Atg12 for 24 hours after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. (f) Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24 hours after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Arrows indicate punctate Atg12 structures in control cells. Quantification of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 in percent are given in the bar graph. ***P<0.0001.