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Video # 1: Enlargement of vesicles after photolysis of the calcium chelator. EGFP-Rab11 Q70L transfected K562 cells were loaded with a permeable photosensible calcium chelator (EGTA-NP-AM) for 30 minutes at 37°C in the dark. Cells were washed and further incubated for two hours with 7 μM MON in the dark. Afterwards, cells were mounted on a polylysine-coated coverslip and placed at 37° onto a chamber for video microscopy and then exposed to UV illumination for 1 minute to destroy the chelator and release the calcium. We have considered time 0 as the first image taken after photolysis. Every 25 seconds, a stack of five z-planes (step 1 μm) were acquired with the green filter set for a 36 min period. Cells were visualized in a Leica DM IRBE epifluorescence microscopy controlled by Metamorph software (Universal Imaging) and with a cooled CCD camera (MicroMax, Pinceton Instruments Inc.).

Video #2: Accumulation of Rab11 at the docking site. E GFP-Rab11 Q70L transfected K562 cells were loaded with a permeable photosensible calcium chelator (EGTA-NP-AM) for 30 minutes at 37°C in the dark. Cells were washed and further incubated for two hours with 7 μM MON in the dark. Afterwards, cells were mounted on a polylysine-coated coverslip and placed at 37° onto a chamber for video microscopy and then exposed to UV illumination for 1 minute to destroy the chelator and release the calcium. Cells were visualized in a Leica DM IRBE epifluorescence microscopy and images were taken at 40 min after photolysis (time 0) for a total period of 22 minutes.

Video # 3: Tubular projections are generated from the enlarged MVBs. EGFP-Rab11 Q70L transfected K562 cells were loaded with a permeable photosensible calcium chelator (EGTA-NP-AM) for 30 minutes at 37°C in the dark. Cells were washed and further incubated for two hours with 7 μM MON in the dark. Afterwards, cells were mounted on a polylysine-coated coverslip, placed at 37° onto a chamber for video microscopy and then exposed to UV illumination for 1 minute to destroy the chelator and release the calcium. Images (30 slides every 11 seconds) were taken by confocal microscopy at 30 min after photolysis (time 0) for a total period of 5 minutes.

Figure S1 : 3D reconstruction of a K562 cell overexpressing Rab11Q70L. Stably transfected K562 cells overexpressing Rab11Q70L were incubated as indicated in Figure 1 and visualized by confocal microscopy. Images for 3D reconstruction were taken using a Nikon Confocal C1 equipped with a 100 × 1.4 NA oil immersion objective and processed with the program EZ-C1. The image clearly shows enlarged MVBs some of them connected by Rab11-decorated tubules.

Figure S2: MON increases exosome release in Rab11 overexpressing K562 cells. Stably Rab11-transfected cells were incubated with 7 μM MON for 12 hours and the exosomal fraction was obtained as described in Materials and Methods. Exosome release was quantified by Western blot. Antibodies against transferrin receptors (TfR) were used as primary antibodies followed by alkaline phosphatase conjugated antibodies. NBT (nitro blue tetrazolium) and BCIP (bromochloroindolyl phosphate) were used as chromagen reagents. Densitometric quantification of the bands obtained was depicted.

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tra_333_sm_6_2_figureS1.avi19653KSupporting info item
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tra_333_sm_6_2_video1.mov6984KSupporting info item
tra_333_sm_6_2_video2.mov1741KSupporting info item
tra_333_sm_6_2_video3.avi23043KSupporting info item

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