Figure 5
Figure 5. PI3K/AKT pathway induces an accumulation of cells in the S phase of cell cycle and cooperates with Stat5 to promote cell growth. (A) Cells (606HS2 and 931HS2) were plated at 1 × 105 cells/mL and cultured for 48 hours in the absence (−; ETOH) or in the presence of indicated concentrations of LY294002 (LY) or NVP-BEZ235 (BEZ). The number of living cells, the percentage of dead cells (trypan blue exclusion), and the percentage of apoptotic cells (by flow cytometry using anti-active caspase 3 Abs) were determined. Bar is the mean ± SEM (n = 3). Statistical differences from the value of the living cells control (−) are indicated as follows: ***P < .001 (by Student t test). Mortality after LY294002 or NVP-BEZ235 treatment was not significantly different (P > .05 by Student t test). (B) Representative cell-cycle distribution of HS2 cells treated or not (−; ETOH) with LY294002 (10μM) or NVP-BEZ235 (120nM) for 24 hours. The percentages of living cells in the G0/G1, S, and G2/M phases of the cell cycle are indicated below. The values are from 1 of 3 independent experiments performed at 24 and 48 hours that gave similar results. (C) Whole-cell extracts from 606HS2 and 931HS2 cells transducing Stat5-sh-RNA17 or Stat5-shRNA19 or control ns-shRNA were analyzed by immunoblotting with the indicated Abs. (D) Lysates from 606HS2 and 931HS2 cells treated or not (−; ETOH) with LY294002 (10μM) or NVP-BEZ235 (120nM) for 4 hours were analyzed by immunoblotting with the indicated Abs. (E) Cells (606HS2 and 931HS2) transducing Stat5-shRNA-17 or control ns-shRNA were plated 72 hours after infection at 1 × 105 cells/mL and cultured in the absence (V, vehicle) or in the presence of LY294002 (10μM). The number of viable cells was scored at 24 and 48 hours. Data are means ± SEM (n = 3). Statistical differences from the value of living cells control (sh-ns + V) are P < .001. Statistical differences between the value of living cells (sh-ns + LY) and (sh17 + LY) and between the value of living cells (sh17 + V) and (sh17 + LY) were P < .001 (by Student t test).

PI3K/AKT pathway induces an accumulation of cells in the S phase of cell cycle and cooperates with Stat5 to promote cell growth. (A) Cells (606HS2 and 931HS2) were plated at 1 × 105 cells/mL and cultured for 48 hours in the absence (−; ETOH) or in the presence of indicated concentrations of LY294002 (LY) or NVP-BEZ235 (BEZ). The number of living cells, the percentage of dead cells (trypan blue exclusion), and the percentage of apoptotic cells (by flow cytometry using anti-active caspase 3 Abs) were determined. Bar is the mean ± SEM (n = 3). Statistical differences from the value of the living cells control (−) are indicated as follows: ***P < .001 (by Student t test). Mortality after LY294002 or NVP-BEZ235 treatment was not significantly different (P > .05 by Student t test). (B) Representative cell-cycle distribution of HS2 cells treated or not (−; ETOH) with LY294002 (10μM) or NVP-BEZ235 (120nM) for 24 hours. The percentages of living cells in the G0/G1, S, and G2/M phases of the cell cycle are indicated below. The values are from 1 of 3 independent experiments performed at 24 and 48 hours that gave similar results. (C) Whole-cell extracts from 606HS2 and 931HS2 cells transducing Stat5-sh-RNA17 or Stat5-shRNA19 or control ns-shRNA were analyzed by immunoblotting with the indicated Abs. (D) Lysates from 606HS2 and 931HS2 cells treated or not (−; ETOH) with LY294002 (10μM) or NVP-BEZ235 (120nM) for 4 hours were analyzed by immunoblotting with the indicated Abs. (E) Cells (606HS2 and 931HS2) transducing Stat5-shRNA-17 or control ns-shRNA were plated 72 hours after infection at 1 × 105 cells/mL and cultured in the absence (V, vehicle) or in the presence of LY294002 (10μM). The number of viable cells was scored at 24 and 48 hours. Data are means ± SEM (n = 3). Statistical differences from the value of living cells control (sh-ns + V) are P < .001. Statistical differences between the value of living cells (sh-ns + LY) and (sh17 + LY) and between the value of living cells (sh17 + V) and (sh17 + LY) were P < .001 (by Student t test).

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