JCS03153 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Association of GFP-PIKfyve with the early endosome requires the activity of a wortmannin-sensitive PI 3-kinase. HeLa cells were transiently transfected with GFP-PIKfyve and after 24 hours incubation a selected cell was imaged live at a capture rate of a frame every 5 seconds. Immediately after capture of the first image (time=0) wortmannin, at a final concentration of 100 nM, was added. For clarity only images captured at 0 and 120 seconds are shown. Data are representative of more than 10 cells imaged under similar conditions. Bar, 10 μm.

• Supplemental Figure 2 -

  Fig. S2. Suppression of PIKfyve does not affect endosomal sorting of the transferrin receptor. HeLa cells were treated with either control, PIKfyve specific siRNA duplex II or a combination of PIKfyve specific siRNA duplexes II and V for 72 hours. Cells were serum starved for 3 hours and labelled with 1 kBq per well ¹²⁵I-transferrin for 60 minutes at 37°C. Cells were chased into 50 μg/ml cold transferrin-containing media for various times at 37°C. Recycled, degraded and internalised fractions were subjected to gamma counting.

• Supplemental Table 1 (Powerpoint) -

  Table S1. Table of the five individual siRNA duplexes used in this study to suppress expression of the human PIKfyve.