Supplemental Material

This article contains the following supporting material:

• Movie 1 - HEp-2 cells control.

  Membrane dynamic of HEp-2 cells, using a 20x lens (recorded 6 h at 1 frame/30 s and played at 1 frame per 1/30 s).

• Movie 2 - CNF1 intoxication of HEp-2 cells.

  Cell morphological changes induced by CNF1 were investigated on HEp-2 cells incubated for 6 h with 1Õ⁹ M, using a 20x lens (recorded at 1 frame/30 s and played at 1 frame per 1/30 s).

• Movie 3 - Vero cells control.

  Membrane dynamic of Vero cells, using a 20x lens (recorded 6 h at 1 frame/30 s and played at 1 frame per 1/30 s).

• Movie 4 - CNF1 intoxication of Vero cells.

  Cell morphological changes induced by CNF1 were investigated on Vero cells incubated for 6 h with 1Õ⁹ M, using a 20x lens (recorded at 1 frame/30 s and played at 1 frame per 1/30 s).

• Movie 5 - HEK293 cells control.

  Membrane dynamic of 293 cells, using a 20x lens (recorded 6 h at 1 frame/30 s and played at 1 frame per 1/30 s).

• Movie 6 - CNF1 intoxication of HEK293 cells.

  Cell morphological changes induced by CNF1 were investigated on 293 cells incubated for 6 h with 1Õ⁹ M, using a 20x lens (recorded at 1 frame/30 s and played at 1 frame per 1/30 s).

• Figure 1 - Smurf1 expression in Smurf1-deficient MEF cells restorates RhoA ubiquitylation

  (A) Immunoblots anti-HA showing RhoA ubiquitylation. Smurf1-deficient MEF cells co-transfected 8h with expression plasmids of histidine-tagged ubiquitin (His-Ub), HA-RhoAL63 with either pEYFP or pYFP-Flag-Smurf1, as indicated. Cells were processed for histidine-tag purification after HA-Rho protein levels normalization. His-Ub crosslinked forms of HA-RhoAL63 were visualized by immunoblotting anti-HA. An anti-HA immunoblot was performed in parallel on 2% of the total lysates to compare the amounts of total HA-RhoAL63 used in the His-Ub purification (labelled Total HA-RhoA). YFP-Flag-Smurf1 expression was verified on immunoblot anti-GFP.

• Figure 2 - In vivo association of Smurf1 with RhoA wild-type and mutants

  (A) Immunoblots showing the association of Smurf1 with RhoA protein mutants. HEK293 cells were co-transfected with an expression plasmid of the HECT-domain catalytic inactive mutant of Smurf1-C699A (Flag-Smurf1-CA) together with Wild-type (RhoAWT), dominant positive (RhoAL63) and dominant negative (RhoAN19) mutants of RhoA. Cells were lyzed and processed for GST purification. GST-protein bound to the beads were resolved on SDS-PAGE and revealed for Flag-Smurf1-CA (anti-Flag), or GST-RhoA proteins (anti-GST). Equal levels of Flag-Smurf1-CA expression in the different conditions were assessed on 2% of the total cell lysates by immunoblotting anti-Flag (labelled Total-Smurf1-CA).

• Figure 3 - Actin cytoskeleton reorganization and Rho protein depletion induced by CNF1 in cell lines

  (A) Immunoblots showing the kinetics of CNF1-induced Rho, Rac and Cdc42 depletion in SW620, MCF7 and T24 cell lines. Cells were treated for different time periods with 10^-9M CNF1. Cells were lysed and immunoblots were performed on 40μg of the total protein lysate. Immunoblots anti-actin were used to evaluate equal loading. (B) Cells were fixed and processed for actin cytoskeleton labeling using TRITC-phalloidin. Bars equal 10 μm