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tra12139-sup-0001-FigureS1.docWord document258KFigure S1: Individual MVBs contain a range of ILV sizes. HeLa cells were incubated with or without EGF for 25 min at 37°C and MVBs subdivided according to the presence of exclusively small (<40 nm) or large (>40 nm) ILVs or both. Most MVBs contain both small and large ILVs in resting cells but some contain exclusively small ILVs and this population is lost after EGF stimulation. EGF stimulation also gives rise to increased numbers of MVBs containing exclusively large ILVs (A). Individual ILV diameters for individual representative MVBs in resting and EGF-stimulated cells are shown in (B).
tra12139-sup-0002-FigureS2.docWord document458KFigure S2: High-pressure freezing confirms that MVBs in Hrs-depleted cells contain ILVs with reduced diameters. Quantitation of high-pressure frozen cells, representative images of which are shown in Figure 6. This data shows that ILV sizes are similar in high-pressure frozen specimens to those of chemically fixed preparations and so the ‘small’ ILVs are not an artefact of chemical fixation.
tra12139-sup-0003-FigureS3.docWord document469KFigure S3: Accumulation of LDL-derived cholesterol in Hrs-depleted cells. Confirming as previously shown by Du et al. [38] that Hrs depletion causes accumulation of cholesterol in endosomes. This raised the possibility that the ‘small’ ILVs might arise because of accumulation of LDL-derived cholesterol. This possibility was excluded in Figure 7.
tra12139-sup-0004-FigureS4.docWord document113KFigure S4: Small ILVs are generated in resting conditions and their formation is dependent on CD63. The number of small (<40 nm) or large (>40 nm) ILVs in unstimulated control, Hrs-depleted or CD63-depleted cells were analysed. Small ILVs do form in resting conditions and their number is decreased following CD63 depletion.
tra12139-sup-0005-FigureS5.docWord document76KFigure S5: Simultaneous depletion of Hrs and CD63 prevents the formation of small ILVs. The effects of depleting Hrs, CD63 or both were again analysed, using separate oligos to those used in Figure 9. The oligos used were: 5′-AAC GTC TTT CCA GAA TTC AAA-3′ (Hrs) [23], and 5′-GGA TGC AGG CAG ATT TTA ATT-3′ (CD63) [39].

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