chemiluminescence images using an image acquisition Chemi- Smart 2000 system (Vilber Lourmat) and Chemi Capt software. Supporting Information Figure S1 Expression of E-cadherin and N-cadherin in nontransformed IAR-2 cells and transformed IAR-6-1, and IAR1170 cells. Total cell lysates were resolved by means of SDS- PAGE and immunoblotted for E-cadherin (A), N-cadherin (B), and a-tubulin (as loading control). Nontransformed IAR-2 cells express epithelial E-cadherin. Transformed IAR-6-1 cells express E-cadherin and do not express N-cadherin. Trans- formed IAR1170 cells retain E-cadherin and also express N- cadherin. Found at: doi:10.1371/journal.pone.0008027.s001 (1.05 MB TIF) Figure S2 Tumorigenic effects of the injection of transformed IAR-1170 cells (clone H5). Cells were injected subcutaneously into athymic nude mice. (A) Tumor volume was measured as described in Materials and Methods. (B) Tumor weight was determined at 4 weeks postinjection, when animals were sacrificed. Each value is the mean6SEM of eight determinations. Tumors were not formed after injection of nontransformed IAR-2 cells. Found at: doi:10.1371/journal.pone.0008027.s002 (2.18 MB TIF) Figure S3 Kymographs generated at the free edges of contacting cells (left) were used to quantifiy the rates of lamellipodial protrusion and retraction (right). In nontransformed IAR-2 cells, the establishment of cell-cell contacts resulted in the decrease of the rates of lamellipodial protrusion at the free edges (from 4.0 mm/min to 2.6 mm/min) (***, p,0.05, t-test). In transformed IAR-6-1 cells, the rates of lamellipodial protrusion and retraction at the free edges of contacting cells did not change within the time of observation. White line on kymograph indicates the time of the establishment of the contact. Data are presented as means6SEM. Found at: doi:10.1371/journal.pone.0008027.s003 (1.40 MB TIF) Figure S4 Schematic representation of the distance map method for measurement of the rate of AJ movement. (A and B) Fragments of confocal images of AJs at times i and i +3 min (image size, 34 mm). Images were acquired at 3-min intervals. (C and D) Skeleton images corresponding to images A and B. (E) Map of the distances from the skeleton at time i. (F) Result of the multiplication of images D and E. The intensity of each pixel in image F corresponds to the distance in pixels traveled by AJs during 3 min. Found at: doi:10.1371/journal.pone.0008027.s004 (0.43 MB PDF) Figure S5 In the presence of Y-27632, the establishment of the contact between IAR-2 cells is not accompanied by contact paralysis. Left: kymograph generated at the site of the cell-cell contact. White line indicates the time of the establishment of the contact. Right: quantification of the rates of lamellipodial protrusion and retraction at the sites of cell-cell contacts. Found at: doi:10.1371/journal.pone.0008027.s005 (0.46 MB PDF) Video S1 Stable cell-cell contacts in an IAR-2 island. Cell-cell contacts are stable during the entire period of observation (Re: Figure 2A). Images were acquired at 5-min intervals for 195 min. Found at: doi:10.1371/journal.pone.0008027.s006 (1.17 MB MOV) Video S2 Cell-cell interactions in a sparse culture of transformed IAR-6-1 cells. Cells extend lamellipodia at different sites along free edges and establish contacts with different neighboring cells. Cell- Adhesive Interactions cell contacts are unstable and often break. Cells move in different directions (Re: Figure 2B). Images were acquired at 5-min intervals for 360 min. Found at: doi:10.1371/journal.pone.0008027.s007 (2.63 MB MOV) Video S3 Cell-cell interactions in a sparse culture of transformed IAR1170 cells (clone H5). The cells establish dynamic contacts with neighboring cells. Cell-cell contacts are unstable and may break (Re: Figure 2C). Images were acquired at 5-min intervals for 425 min. Found at: doi:10.1371/journal.pone.0008027.s008 (3.42 MB MOV) Video S4 Dynamics of cell-cell interactions between nontrans- formed IAR-2 cells in a narrow wound. By 15-20 min, protrusive activity at the contact site is inhibited (contact paralysis). At the same time, the extension of lamellipodia at the free edges of contacting cells decreases (Re: Figure 4A). Images were acquired at 3-s intervals for 20 min. Found at: doi:10.1371/journal.pone.0008027.s009 (10.15 MB MOV) Video S5 Dynamics of cell-cell interactions between transformed IAR-6-1 cells in a narrow wound. Upon formation of the contact, protrusions extend both at the site of the contact (absence of contact paralysis) and at the free edges (Re: Figure 4C). Images were acquired at 15-s intervals for 28 min. Found at: doi:10.1371/journal.pone.0008027.s010 (4.28 MB MOV) Video S6 GFP-E-cadherin dynamics at the sites of cell-cell contacts in a sparse IAR-2 culture. Nontransformed IAR-2 cells were stably transfected with GFP-E-cadherin. GFP-E-cadherin accumulates in the zones of cell-cell interactions, forming stable tangential AJs (Re: Figure 5A). Images were acquired at 10-min intervals for 170 min. Found at: doi:10.1371/journal.pone.0008027.s011 (0.27 MB MOV) Video S7 Remodeling of AJs in transformed IAR-6-1 cells. Transformed IAR-6-1 cells were stably transfected with GFP-E- cadherin. Upon establishment of cell-cell contact, E-cadherin aggregates in dot-like AJs at the site of the contact. AJs can grow, rearrange, and relocate in the area of the contacts. The disruption of AJs results in detachment of one cell from another (Re: Figure 5B). Images were acquired at 10-min intervals for 320 min. Found at: doi:10.1371/journal.pone.0008027.s012 (0.64 MB MOV) Video S8 Accumulation of GFP-E-cadherin at the contact between two nontransformed IAR-2 cells in a narrow wound. During the formation of the cell-cell contact, GFP-E-cadherin accumulates in a tangential line at the boundary between two cells (Re: Figure 6A). Images were acquired at 30-s intervals for 13 min. Found at: doi:10.1371/journal.pone.0008027.s013 (0.81 MB MOV) Video S9 GFP-E-cadherin dynamics at the site of the cell-cell contact of transformed IAR-6-1 cells in a narrow wound. GFP-E- cadherin initially accumulates in small, dot-like clusters. A majority of E-cadherin dots transform into radial strands. Within the time of observation, the size of radial AJs can grow (Re: Figure 6B). Images were acquired at 30-s intervals for 47 min. Found at: doi:10.1371/journal.pone.0008027.s014 (0.98 MB MOV) PLoS ONE | www.plosone.org 16 November 2009 | Volume 4 | Issue 11 | e8027