FIGURE 9.

FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN^+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN^+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 10⁴ of FoxO3a^+/+ and FoxO3a^−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a^−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 10⁴ of Foxo3a^−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a^−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a^−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.