JCS03238 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Co-localization of Bds and Dex with TfR; lack of co-localization with Lamp-1 and with the endoplasmic reticulum and MHCII-rich vesicles. Conditions of the experiments are as in Fig. 1E,F: 10 minutes of incubation of DCs with Bds or Dex, then fixation followed by permeabilization and immunolabelling with specific antibodies. Panels A and C show the low level of co-localization of Bds and Dex, respectively, with TfR; panels B and D show the lack of co-localization of the tracers with Lamp-1; panels E and F show the lack of co-localization of Bds with the ER marker, calreticulin, and with the MHCII-rich vesicle marker, HLA-DR, respectively. Bar, 10 μm (E), also valid for all panels.

• Supplemental Figure 2 -

  Fig. S2. DCs exposed to tracers for various times and immunolabelled for rabankyrin-5, EEA1 and transferrin. (A,B) DCs exposed to either Bds or Dex and then analyzed quantitatively as in Fig. 2 for the (A) Bds, Tf and TfR, Dex, (B) Rbk and EEA1. (C) Data in B with representative images. (D,E) Representative images corresponding to the quantitative panels B and C of Fig. 2. In this case the cells, loaded with FITC-Bds for the times indicated, were fixed and dually immunolabelled for EEA1 (all panels, blue), together with (red) either Rbk-5 (D) or Tf (E). The top line of C-E shows the merges of their triple-labelled images.

• Supplemental Figure 3 -

  Fig. S3. Endocytosis of transferrin: inhibition by Wort and no effect of amiloride. DCs were preincubated with Wort (A) or amiloride (B) as in Fig. 3 and then exposed to TRITC-transferrin for 10 minutes.

• Supplemental Figure 4 -

  Fig. S4. Akt phosphorylation in DCs exposed to PIP3. Panels A and B show that the phosphoryl-akt signal (red) is hardly appreciable in DCs, at rest (A) and also 10 minutes after Bd (green) application (B). By contrast, the signal is clearly visible in many DCs exposed to PIP3, non-pretreated (C), preloaded with BAPTA (D) or pretreated with Wort (E).

• Movie 1
• Movie 2 -

  Movies 1 and 2. Rotating 3D reconstructions from DCs pretreated with Wort (A) or preloaded with BAPTA (B), exposed first to PIP3 (20 minutes) and then to the beads (10 minutes), fixed and surface immunolabelled for d/A. The tridimensional DCs were reconstituted from stacks of merged images. In both panels the large Bd-rich vacuoles (green), assembled in PIP3-treated cells in spite of the inhibitory pretreatments with Wort or BAPTA, are distributed primarily in the proximity of the plasma membrane but do not coincide with the d/A-positive surface patches (red) resulting from the exocytosis of enlargeosomes.