JCS02939 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Treatment of DC with calpain inhibitors results in accumulation of paxillin in podosomes. DC plated overnight on poly-L-lysine coated glass coverslips were left untreated (panels A,B,C) or were treated with ALLM (panels D,E,F) or ALLN (panels G,H,I) and then fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and stained to detect paxillin and actin. Inhibition of calpain resulted in enhanced accumulation of paxillin and actin in DC podosomes. The micrographs are representative of the cytoskeletal organisation of DC detected in three independent experiments. Bar, 10 μm.

• Supplemental Figure 2 -

  Fig. S2. Treatment of DC with cathepsin B and proteosome inhibitors result in podosome loss. DC plated overnight on poly-L-lysine coated glass coverslips were treated with proteosome inhibitor (A-C) or cathepsins inhibitor (D-E) or and fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and stained to detect vinculin (green, panels A and D) and actin (red, panels B and E). Merged images are shown in panels C and F. Treatment of DC with cathepsins and proteasome inhibitors resulted in disassembly of podosomes. Bar, 10 μm.

• Supplemental Figure 3 -

  Fig. S3. Podosomes occupy the most prominent area of adhesion of DCs to the substratum. Confocal micrographs of DCs plated overnight on poly-L-lysine coated glass coverslips showing the distribution of vinculin in green (A), actin in red (B) and of the adhesion points of the cell on the substratum as detected by IRM (C). DCs form podosome clusters behind the leading edge that account for the major area adhesion of DC on the substratum. DCs can also form focal complexes detected as punctuated clusters of vinculin (asterisks) or focal adhesion like structures detected as clusters of vinculin forming rods (arrow heads). Bar, 10 μm.

• Supplemental Figure 4 -

  Fig. S4. In vitro incubation of WASP with calpain results in WASP cleavage. We obtained FLAG-WASP protein using Promega in vitro translation system and incubated the obtained protein preparations in the presence of absence of increasing amounts of porcine calpain 2 (Calbiochem) for 30 minutes. Protein preparations were subjected to SDS-PAGE and Western blotted. Immunodetection using an anti-FLAG antibody shows the cleavage of WASP by calpain. Asterisks mark WASP calpain cleavage products with the same molecular weight as the ones detected in DC lysates.

• Movie 1 -

  Movie 1. Interference reflection microscopy films of untreated spleen derived SV129 DC plated on poly-L-lysine-coated glass viewing chambers. Micrographs were taken 10 seconds apart and displayed at 10 frames per second.

• Movie 2 -

  Movie 2. Interference reflection microscopy films of ALLM treated spleen derived SV129 DC plated on poly-L-lysine-coated glass viewing chambers. Micrographs were taken 10 seconds apart and displayed at 10 frames per second. Treatment of DCs with ALLM resulted in stabilisation of podosomes.

• Movie 3 -

  Movie 3. Phase-contrast films of untreated spleen derived SV129 DC plated on poly-L-lysine-coated Petri dishes. Micrographs were taken 5 minutes apart and displayed at 10 frames per second. DCs.

• Movie 4 -

  Movie 4. Phase-contrast films of ALLM treated spleen derived SV129 DC plated on poly-L-lysine-coated Petri dishes. Micrographs were taken 5 minutes apart and displayed at 10 frames per second. DCs.

• Movie 5 -

  Movie 5. Interference reflection microscopy films of untreated spleen derived WASP null DC expressing eGFP-WASP plated on poly-L-lysine-coated glass viewing chambers. Micrographs 20 seconds apart were taken using confocal microscopy and displayed at 10 frames per second.

• Movie 6 -

  Movie 6. Confocal microscopy films of untreated spleen derived WASP null DC expressing eGFP-WASP plated on poly-L-lysine-coated glass viewing chambers. Micrographs were taken 20 seconds apart and displayed at 10 frames per second.

• Movie 7 -

  Movie 7. Interference reflection microscopy films of ALLM treated spleen derived WASP null DC expressing eGFP-WASP plated on poly-L-lysine-coated glass viewing chambers. Micrographs 20 seconds apart were taken using confocal microscopy and displayed at 10 frames per second.

• Movie 8 -

  Movie 8. Confocal microscopy films of ALLM treated spleen derived WASP null DC expressing eGFP-WASP plated on poly-L-lysine-coated glass viewing chambers. Micrographs were taken 20 seconds apart and displayed at 10 frames per second.