Fig. 5. Roles of E-cadherin and PI3-K in TGFß responsiveness and effect of suprabasal 6ß4 on basal cell proliferation. (A) Stratified wt and untransduced Inv6ß4 keratinocytes were compared with Inv6ß4 cells transduced with dnEcad or dnEcadC25 retroviral vectors following treatment with 2 ng/ml TGFß1 for 1 hour. *Significantly different from untransduced Inv6ß4 keratinocytes, P<0.05. (B) Differentiated Inv6ß4 cells were plated onto monolayers of wt cells overnight, incubated with the inhibitors shown or DMSO alone, then treated with 2 ng/ml TGFß1 for 1 hour. (C) Western blot of total and activated (pAkt) Akt levels in wt and transgenic stratified keratinocytes, ±LY 294002. Actin is the loading control. (D) BrdU incorporation in keratinocyte monolayers (basal, left-hand panel) or in stratified cultures formed by combining suspended wt or Inv6ß4 cells with wt monolayers (suprabasal, right-hand panel). Cells were serum starved for 24 hours, then treated with complete medium - (black bars) or + (grey bars) 2 ng/ml TGFß1 for 20 hours. (A,B,D) Each data set represents the average count of % Smad2/3-positive (A,B) or BrdU-positive (D) nuclei versus total Hoescht-stained nuclei from three separate fields (monolayers: 100-200 cells/field; recombined and post-confluent cultures: 400-500 cells/field).