JCS03218 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 -

  Adobe PDF

• Supplemental Figure 1 -

  Fig S1. Confocal immunofluorescence analysis of E-cadherin, cytokeratin 8, fibronectin and vimentin expression in MDCK cell transfectants. Cells were labelled with specific antibodies against E-cadherin, cytokeratin 8, fibronectin and vimentin. Nuclei were stained with DAPI.

• Supplemental Figure 2 -

  Fig S2. Podoplanin induces actin reorganization which depends on both the ectodomain and the cytoplasmic tail. F-actin was revealed by staining with Alexa Fluor 594-labelled phalloidin, and vinculin was detected by staining with a specific antibody. Nuclei were stained with DAPI. All panels show confocal images.

• Supplemental Figure 3 -

  Fig. S3. The colocalization of EGFP-tagged PWT, PΔEC and PΔCT proteins with RhoA and Rac1 in MDCK cell transfectants was analyzed by confocal immunofluorescence microscopy. Panels show a single confocal microscopy section. Insets show magnification of the tip of a cell protrusion where colocalization of PWT and RhoA can be observed.

• Movie 1 -

  Movie 1. Migratory behaviour of MDCK cell transfectants during in vitro wound healing. See Fig. 5A. Images were taken every 10 minutes during 12 hours and displayed at 5 frames/second.

• Movie 2 -

  Movie 2. Dynamics of podoplanin subcellular localization in a MDCK cell transfected with EGFP-tagged PWT. Arrows indicate accumulation of podoplanin at ruffles. Arrowheads mark intracellular vesicles containing podoplanin. Images were taken every 3 seconds for 10 minutes and are displayed at 5 frames/second.

• Movie 3 -

  Movie 3. Dynamics of podoplanin subcellular localization in a migrating MDCK cell expressing EGFP-tagged PWT. See Fig. 6. Arrows indicate the direction of cell migration. Arrowheads mark the clustering of podoplanin at different plasma membrane locations. Images were taken every 10 minutes and are displayed at 5 frames/second.

• Movie 4 -

  Movie 4. Dynamics of PΔCT subcellular localization during migration of a MDCK cell expressing EGFP-tagged PΔCT. See Fig. 6. Details are as in the legend of Movie 3. Note that lamellipodial and membrane ruffling activities are less intense in this migrating epithelial cell when compared with the cell showed in Movie 3.

• Movie 5 -

  Movie 5. Dynamics of PΔEC subcellular localization during migration of a MDCK cell expressing EGFP-tagged PΔEC. See Fig. 6. Details are as in the legend of Movie 3. Note perinuclear accumulation of the truncated protein. Note also that migration of this cell is not as directional as the cells showed in Movies 3 and 4.