Fig. 2. (a) COS-7 cells were transiently transfected with empty vector (Control), DN-Rab5, CA-Rab5 or WT-Rab5 and GFP-LC3 or mRFP-LC3 (3:1 ratio) for 24 hours. GFP-positive or mRFP-positive cells with increased numbers of LC3-positive vesicles (>20 vesicles per cell) were counted. 29% of control cells had >20 vesicles per cell. ^***P<0.0001. (b) Western blot analysis of COS-7 cells co-transfected with empty vector (C), DN-Rab5, CA-Rab5 or WT-Rab5 and Myc-LC3 for 24 hours in the presence of 200 nM bafilomycin A1 (treated for last 15 hours), using anti-Myc antibody. GFP was used as a transfection control. Representative image from three independent experiments; quantification of the band intensities from these experiments represented as LC3-II:GFP ratio is shown in the graph; ^*P<0.05. (c) Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector [either untreated or treated for 24 hours with 10 mM 3-methyladenine (3-MA)] or with dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. An increased abundance of large punctate Atg5 structures can be noticed with DN-Rab5 and 3-MA treatment. (d) HeLa cells were transfected with siRNA targeting Vps34 or control siRNA for 48 hours after which GFP-Atg5 together with siRNA was transfected for further 24 hours. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of Q74-expressing HeLa cells with aggregates is shown in the graph. Cells were transiently transfected with control siRNA or siRNA targeting Vps34 for 48 hours and with HA-tagged huntingtin exon 1 containing 74 polyglutamine repeats for further 24 hours. ^***P<0.0001. (e) Colocalisation of GFP-Atg5 structures (green) with Myc-tagged FYVE (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Myc-FYVE for 24 hours. (f) Colocalisation of GFP-Atg5 structures (green) with beclin 1 (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Flag-tagged wild-type (WT) beclin 1. (g) Colocalisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15 hours. In panels e-g we observed >30% colocalisation between GFP-Atg5 structures and saponin-extracted, membrane-associated, FYVE, beclin 1 or Rab5 in cells that expressed both of the respective proteins. (h) Colocalisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells co-transfected with DN-Rab5 and GFP-Atg5 and HA-tagged Atg12 for 24 hours. Nuclei labelled with DAPI are in blue.