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. Author manuscript; available in PMC: 2009 Feb 3.

Published in final edited form as: J Cell Sci. 2008 Apr 22;121(Pt 10):1649–1660. doi: 10.1242/jcs.025726

a. COS-7 cells were transiently transfected with empty vector (Control), DN-, CA- or WT-Rab5 and GFP-LC3/mRFP-LC3 (3:1 ratio) for 24h. GFP-/mRFP-positive cells with increased LC3-positive vesicles (> 20 vesicles per cell) were quantified. The proportion of control cells with >20 vesicles per cell is 29%. *** - P < 0.0001. b. Western blot analysis of COS-7 cells transfected with empty vector (C), DN-, CA- or WT-Rab5 and myc-LC3 for 24h in the presence of 200nM Bafilomycin A1 (treated for last 15h), with anti-myc antibody. GFP was used as a transfection control. Quantitation of the band intensities from 3 independent experiments is shown * - P < 0.05. c. Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector (either untreated or treated for 24h with 10mM 3-methyladenine (3-MA)) or dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. d. HeLa cells were transfected with siRNA against Vps34 or a control siRNA for 48h after which GFP-Atg5 together with siRNA was transfected for further 24h. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of proportions of GFP-expressing cells with Q74 aggregates in HeLa cells transiently transfected with siRNA for control or Vps34 for 48h and further with HA-tagged huntingtin exon-1 with 74 polyQ repeats for the next 24h. *** - P < 0.0001. e. Co-localisation of GFP-Atg5 structures (green) with myc-tagged FYVE (red) in Hela cells transfected with DN-Rab5 together with GFP-Atg5 and myc-FYVE for 24h. f. Co-localisation of GFP-Atg5 structures (green) with Beclin-1 (red) in Hela cells transfected with DN-Rab5 together with GFP-Atg5 and Flag-tagged wild-type (WT) Beclin-1. g. Co-localisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15h. In panels e-g we observed more than 30% co-localisation between GFP-Atg5 structures and saponin extracted, membrane associated, FYVE, Beclin-1 or Rab5 in cells expressing both the relevant proteins. h. Co-localisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells transfected with DN-Rab5 together with GFP-Atg5 and HA-tagged Atg12 for 24h. Nuclei labelled with DAPI are in blue.

a. COS-7 cells were transiently transfected with empty vector (Control), DN-, CA- or WT-Rab5 and GFP-LC3/mRFP-LC3 (3:1 ratio) for 24h. GFP-/mRFP-positive cells with increased LC3-positive vesicles (> 20 vesicles per cell) were quantified. The proportion of control cells with >20 vesicles per cell is 29%. *** - P < 0.0001. b. Western blot analysis of COS-7 cells transfected with empty vector (C), DN-, CA- or WT-Rab5 and myc-LC3 for 24h in the presence of 200nM Bafilomycin A1 (treated for last 15h), with anti-myc antibody. GFP was used as a transfection control. Quantitation of the band intensities from 3 independent experiments is shown * - P < 0.05. c. Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector (either untreated or treated for 24h with 10mM 3-methyladenine (3-MA)) or dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. d. HeLa cells were transfected with siRNA against Vps34 or a control siRNA for 48h after which GFP-Atg5 together with siRNA was transfected for further 24h. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of proportions of GFP-expressing cells with Q74 aggregates in HeLa cells transiently transfected with siRNA for control or Vps34 for 48h and further with HA-tagged huntingtin exon-1 with 74 polyQ repeats for the next 24h. *** - P < 0.0001. e. Co-localisation of GFP-Atg5 structures (green) with myc-tagged FYVE (red) in Hela cells transfected with DN-Rab5 together with GFP-Atg5 and myc-FYVE for 24h. f. Co-localisation of GFP-Atg5 structures (green) with Beclin-1 (red) in Hela cells transfected with DN-Rab5 together with GFP-Atg5 and Flag-tagged wild-type (WT) Beclin-1. g. Co-localisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15h. In panels e-g we observed more than 30% co-localisation between GFP-Atg5 structures and saponin extracted, membrane associated, FYVE, Beclin-1 or Rab5 in cells expressing both the relevant proteins. h. Co-localisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells transfected with DN-Rab5 together with GFP-Atg5 and HA-tagged Atg12 for 24h. Nuclei labelled with DAPI are in blue.