JCS025726 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Western blot analysis of HeLa cells transfected with control, siRNA targeting Rab5a, Rab5b and Rab5c or siRNA targeting Rab5a, Rab5b and Rab5c simultaneously for 72 hours and probed with antibodies against Rab5a, Rab5b or Rabc. Actin was used as a loading control.

• Supplemental Figure S10 (Adobe PDF) -

  Fig. S10. Analysis of GFP-Atg5 structures in HeLa cells transfected with control vector or dominant-negative dynamin (DN-Dyn) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue.

• Supplemental Figure S11 (Adobe PDF) -

  Fig. S11. HeLa cells transfected with control vector or DN-Vps4B together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg12-Atg5 complex. The ratio of the Atg12-Atg5 complex versus free Atg12 is shown in the graph.

• Supplemental Figure S12 (Adobe PDF) -

  Fig. S12. Lysates from HeLa cells transfected with control or clathrin siRNA for 72 hours were blotted for clathrin and actin as control.

• Supplemental Figure S13 (Adobe PDF) -

  Fig. S13. HeLa cells, treated with or without 5 mM β-cyclodextrin for 24 hours, were assessed by confocal microscopy for the uptake of Alexa-Fluor-594-labeled human transferrin (see Materials and Methods for details). Note that the transferrin is not endocystosed and remains of the surface of the β-cyclodextrin-treated cells.

• Supplemental Figure S14 (Adobe PDF) -

  Fig. S14. Western blot analysis of HeLa cells transfected with control vector (Cont) or DN-dynamin (DN-Dyn) in the presence or absence of bafilomycin A1 (BafA1) for endogenous LC3 and actin. Under exposure conditions that allow endogenous LC3-II quantification in these cells, the LC3-I signal is frequently too low to detect.

• Supplemental Figure S15 (Adobe PDF) -

  Fig. S15. Western blot analysis of HeLa cells transfected with control (Cont) or clathrin (Clat) siRNA in the presence or absence of Bafilomycin A1 (BafA1) for endogenous LC3 and actin.

• Supplemental Figure S16 (Adobe PDF) -

  Figure 16. Lysates from HeLa cells transfected with control vector (Cont) or wild-type Rheb were blotted for phospho- (S6-P) and total- (S6-T) ribosomal protein S6. The increased S6-P levels are indication of increased mTOR activity in Rheb-transfected cells.

• Supplemental Figure S17 (Adobe PDF) -

  Fig. S17. COS-7 cells transfected with HA-tagged huntingtin exon 1 (Q74) for 48 hours were labelled with anti-HA antibody to detect Q74. Q74 expressing cells with aggregates are marked by arrows and an aggregate-containing cell with abnormal nucleus is marked with an arrowhead.

• Supplemental Figure S18 (Adobe PDF) -

  Fig. S18. DAPI-stained nuclei showing abnormal nuclear morphologies are marked by arrows.

• Supplemental Figure S19 (Adobe PDF) -

  Fig. S19. COS-7 cells expressing GFP-LC3 for 24 hours showing (a) <20 vesicles or (b) >20 vesicles.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. COS-7 cells transfected with GFP-Q74 and either pcDNA3.1 (empty vector) or dominant-negative (DN) Rab5 at a ratio of 1:3, were treated with or without 100 µM of the inositol monophosphatase inhibitor L-690,330 for 48 hours post transfection and assessed for the percentage of GFP-positive cells with aggregates.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Analysis of endogenous Atg5 structures in HeLa cells in control cells (untreated or treated with 3MA) or transfected with DN-Rab5. Nuclei labelled with DAPI are in blue.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. HeLa cells transfected with control or hVps34 siRNA for 72 hours were blotted for hVps34 and actin.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. No obvious colocalisation of GFP-Atg5 structures (green) with mRFP-LC3 (red) in HeLa cells transfected with DN-Rab5 together with GFP-Atg5 and mRFP-LC3 for 24 hours.

• Supplemental Figure S6 (Adobe PDF) -

  Figure 6. No colocalisation of GFP-Atg5 structures (green) with the Golgi marker p230 (red) in HeLa cells transfected with GFP-Atg5 in the presence of 3-methyladenine (3-MA).

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. HeLa cells transfected with control or Atg7 siRNA for 72 hours were blotted for Atg7 and actin (as control).

• Supplemental Figure S8 (Adobe PDF) -

  Fig. S8. Colocalisation of GFP-Atg5 structures (green) in Atg7 knockdown cells with beclin 1 (red).

• Supplemental Figure S9 (Adobe PDF) -

  Fig. S9. HeLa cells transfected with control or beclin 1 (Bec-1) siRNA for 72 hours were blotted for beclin 1 and actin.