FIG. 6.

Smad4 is necessary for TGF-β-dependent migration in a scratch assay. (A, B, and C) The Tet-induced silencing of Smad4 in HaCaT-TRS4 cells abolishes TGF-β-induced migration in a scratch assay. HaCaT-TR, HaCaT-TRS4, or HaCaT-TRS4-rescue cells were grown to confluence with or without Tet for 48 h and were then serum starved for 24 h. A scratch was introduced into the confluent cell monolayer, and TGF-β-induced migration was monitored with time-lapse microscopy for 48 h. A photograph of the cells after 48 h of migration is shown in panel A. Tracking of cells at the leading edge of the scratch was performed on the acquired images using the Tracker software for the HaCaT-TR cells (+Tet) and HaCaT-TRS4 cells (±Tet) (B). Mean speeds were calculated from the cell tracking analysis using the Mathematica software (C). The mean speeds of cells in different condition are represented, and the asterisk indicates conditions that are significantly different from the others with a P value of <0.00001. (D) Smad4 is also necessary for TGF-β-induced migration in pancreatic cells. Colo-TR and Colo-TRS4 cells were grown to confluence with or without Tet for 48 h. A scratch was introduced into the monolayer, and TGF-β-induced “wound” closure was photographed after 48 h of cell migration. (E) EGF-induced migration is still functional in the absence of Smad4. HaCaT-TR or HaCaT-TRS4 cells were grown to confluence with or without Tet for 48 h and then serum starved for 24 h. After scratching, cells were incubated with or without TGF-β or EGF for 48 h, at which time the images were taken.