Figure 5. FOXO1 inhibits growth and induces apoptosis in MedB-1 cells.

(A) Expression of FOXO1-ER fusion protein and endogenous FOXO1 in MedB-1 cells transduced with constitutively active FOXO1 variant or with empty vector (EV) was analyzed by immunoblot. (B) MedB-1 cells transduced with FOXO1ER or with empty vector were seeded in six-well plates at a density of 1×10⁵ cells in 3 ml of complete culture medium. The next day EV and FOXO1ER cells were treated with 200 nM 4-OHT or with vehicle. Live cells were count by hemacytometer according to morphological criteria. The data of one of three independent experiments that yielded similar results are shown as mean±SD. All measurements were done in triplicate. (C) FOXO1 induces G₁ arrest. The cell cycle distribution was measured 48 h after treatment with 4-OHT using PI staining. The data are shown as mean±SD of three measurements. Results of one of three independent experiments that yielded similar results are shown (D) Cell were seeded and treated with 4-OHT as it was described for (A). Cell death was measured by annexin V/7-AAD staining simultaneously with cell counting. The specific apoptosis (SA) was calculated as: SA(%) = 100 × (A_E − A_C)/(100 − A_C), where A_E equals the percentage of apoptotic cells in the experiment group and A_C equals percent of apoptotic cells in the control group. The measurements were done in triplicate by using flow cytometry. The results of one of three independent experiments that yielded similar results are shown as mean of SA±SD.