Figure 4. Expression of activated Rho (Q63L) or Rac (Q61L) impairs formation of cell-cell contacts in NBT-II cells.
A) NBT-II cells were transiently transfected with the empty pEGFP vector or plasmids encoding GFP-fusion proteins containing dominant negative/constitutively active Rac (N17/Q61L) or YFP-fusion proteins containing dominant negative/constitutively active Rho (N19/Q63L). Forty-eight hours post-transfection the cells were subjected to a two-hour calcium switch. Expressing cells were identified as GFP positive (left column) and cell-cell contacts were visualized by staining with an E-cadherin antibody and a rhodamine-conjugated secondary antibody (right column). The arrows indicate examples of Rac Q61L and RhoQ63L expressing cells that exhibit loss of cell-cell contacts. B) The proportion of GFP/YFP positive cells containing more than one intercellular contact was quantified. Results shown correspond to at least three independent experiments (+/− S.E.M) with over one hundred GFP/YFP positive cells scored per experiment. The data were analyzed using a one-way analysis of variance (p<0.0005) and the Dunnett’s multiple comparison post test (*, p<0.01).