FIGURE 1.

tTG is localized to the leading edges of actively migrating cells, and its cross-linking activity is necessary for cell migration. A, scratch assays were performed on HeLa cells (top panels) and MDAMB231 cells (bottom panels) treated without (Untreated) or with EGF and without or with MDC as indicated. MDAMB231 cells were fixed 12 h after striking the wound; HeLa cells were fixed after 24 h. The cells were then visualized using light microscopy, and the extent of wound closure was determined. One set of untreated cells was fixed immediately after striking the wound (Untreated 0 h.) to indicate the size of the initial wounds. The widths of the initial wounds are indicated by dashed lines. B, the extracts collected from HeLa cells transfected with control-RNAi, tTG-RNAi 1, or tTG-RNAi 2 were immunoblotted with tTG and actin antibodies (left panels). Scratch assays were then performed on cells transfected with the same siRNAs, treated without (Untreated) or with EGF. The cells were processed as outlined in A (right panels). C and D, duplicate sets of serum-starved cultures of HeLa cells and MDAMB231 cells were treated without (Untreated) or with EGF for increasing lengths of time, as indicated, and then were fixed. C, immunofluorescence was performed on one set of the cells using a tTG antibody, rhodamine-conjugated phalloidin (Actin), and DAPI (to stain nuclei). D, immunofluorescence was performed on the second set of cells using tTG and cortactin antibodies and DAPI. Representative fluorescent images of the cells are shown, and the localization of tTG and cortactin at leading edges is indicated with arrows. E, the extracts collected from MDAMB231 cells transfected with control-RNAi, tTG-RNAi 1, or tTG-RNAi 2 were immunoblotted with tTG and actin antibodies (top panels). Scratch assays were then performed on cells transfected with the same siRNAs, and the cells were processed as outlined in A (bottom panels).