FIG. 5.

Effects of wt and mutant p85 (PI3K) and of wt PTEN expression vectors on HGF/SF-mediated protection of MDCK cells. (A) The regulatory subunit of PI3K (p85) modulates MDCK survival. Subconfluent proliferating cells in 100-mm-diameter dishes were transiently transfected overnight, using Lipofectamine and 10 μg of each vector per dish: empty vector (control), p85(wt), or p85(DN). Cells were washed, subcultured into 96-well dishes, preincubated with or without HGF/SF (100 ng/ml for 48 h), exposed to ADR (15 μM for 2 h), washed three times to remove ADR, postincubated for 72 h in fresh drug-free medium, and assayed for MTT dye conversion. Values of cell viability are based on 10 replicate wells. p85(wt) protected parental and Gab1 (wt) cells against ADR in the absence of HGF/SF (P < 0.001); p85(DN) blocked the HGF/SF-mediated protection of parental cells against ADR (P < 0.001). (B) PTEN blocks HGF/SF-mediated protection of parental MDCK cells. Transient transfection assays were performed as for panel A. For the pooled data from the two dishes for each assay condition, cells treated with empty vector plus HGF/SF and ADR showed a significantly reduced cell viability compared with cells treated with (PTEN, HGF/SF, and ADR) (P < 0.001); there was little or no increase in survival of cells treated with PTEN, HGF/SF, and ADR as compared with PTEN plus ADR.