Volume 8 issue 3 March 2007
Salmonella trafficking is Defined by Continuous Dynamic Interactions with the
Endolysosomal System
D Drecktrah , LA Knodler, D Howe and O Steele-Mortimer

Figure S1. Optimization of conditions for labelling and imaging LE/Lys. (A) Loss of fluid phase dextran-488 after fixation and processing for immunofluorescence microscopy. The panel on the left shows the endocytic pathway labeled with dextran-488 in live cells. The panel on the right shows the same field of view after fixation and processing (see Materials and Methods). Below each panel is a graph of pixel intensity along the red line in the micrographs (arrows at either end) illustrating the loss of signal after processing. (B) To compare internalization protocols for labelling LE/Lys dextran-488 and dextran-568 were internalized into HeLa cells. In all three protocols dextran-488 was internalized for 4 h and then chased to lysosomes by o/n chase. Dextran-568 was (i) co-internalized with dextran-488 for 4 h (to give maximal co-localization), (ii) added immediately after dextran-488 was removed, and internalized overnight then chased for 3 h in dextran-free media or (iii) added for 20 min immediately before imaging (see supplemental Figure S1 for schematic). The percent dextran-568 that colocalized with dextran-488 containing lysosomes was then determined from images acquired during live cell imaging. In separate experiments cells were transfected with LAMP1-GFP then dextran-568 was internalized for either (i) 4 h and chased o/n in dextran-free media, or (ii) o/n and chased in dextran-free media for 3 h before imaging. All values are the mean ± SD from one representative experiment.

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Figure S2. 3-dimensional image volume rendering of SCVs containing Red- Salmonella , (blue) showing content mixing of dextran-647 (red) delivered from lysosomes and dextran-488 (green) from incoming endocytic traffic. HeLa cells, 3 h p.i. See Figure 2B for details.

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Figure S3. Time-lapse series showing accumulation of the lysosomal content marker dextran-488 (green) in a single SCV containing Red- Salmonella (red). Confocal live cell imaging was initiated at 18 min p.i. (0:00) acquiring single optical frames every 3 sec. Play back speed 6 frames per sec. See Figure 2D for details.

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Figure S4. Time-lapse series showing interactions of dextran-488 loaded lysosomes (green) with SCVs containing Red- Salmonella (red) in HeLa cells. Confocal live cell imaging was initiated at 45 min p.i. acquiring single optical frames every 0.7 sec. See Figure 5 for details.

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Figure S5. Time-lapse series showing interactions of dextran-488 loaded lysosomes (green) with vacuoles containing Red- E. coli pInv (red) in HeLa cells. Confocal live cell imaging was initiated at 45 min p.i. acquiring single optical frames every 0.7 sec. See Figure 5 for details.

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Figure S6. 3-dimensional image volume rendering of Salmonella (red) at 6 h p.i. residing in a C. burnetii (green) parasitophorous vacuole stained for LAMP1 (blue) in co-infected HeLa cells. See Figure 6 for details.

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