Figure 3. PIB5PA-triggered inhibition of cell proliferation is due to inhibition of PI3K/Akt.

(a) Mel-FH.PIB5PA and ME1007.PIB5PA cells that carried a lentiviral-based 4-OHT-responsive inducible gene expression system and their parental counterparts were exposed to 4-OHT at indicated concentrations for 24 h. Whole-cell lysates were subjected to western blot analysis. (b) Whole-cell lysates from Mel-FH.PIB5PA and ME1007.PIB5PA cells treated with 4-OHT (10 nM) for 24 h with or without withdrawal of 4-OHT for another 24 h were subjected to western blot analysis. (c) Mel-FH.PIB5PA and ME1007.PIB5PA cells treated with 4-OHT (10 nM) for 24 h with or without withdrawal of 4-OHT for another 48 h were subjected to proliferation assays using the BrdU incorporation method (n=3, mean±s.e.m.). (d) Mel-FH.PIB5PA and ME1007.PIB5PA cells were seeded onto six-well plates (2,000 cells per well) for 24 h before the addition of 4-OHT (10 nM). Seventy-two hours later, 4-OHT was withdrawn from representative wells. Cells were allowed to grow for another 9 days before being fixed with methanol and stained with crystal violet. Scale bar, 1 cm. (e) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transiently transfected with vector alone or a myr-Akt construct. Twenty-four hours later, cells were treated with 4-OHT (10 nM) for a further 24 h. Whole-cell lysates were subjected to western blot analysis. (f) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transiently transfected with vector alone or a myr-Akt construct. Twenty-four hours later, cells were treated with 4-OHT (10 nM) for a further 48 h before cell proliferation was measured by the BrdU incorporation method (n=3, mean±s.e.m.). (g) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transfected with vector alone or a myr-Akt construct. Twenty-four hours later, viable cells (2,000 cells per well in six-well plates) were allowed to grow for a further 12 days before being fixed with methanol and stained with crystal violet. Scale bar, 1 cm.