AKT and ERK kinases inhibitors restore FOXO1 expression in cHL cell lines. (A) Constitutive activation of ERK and AKT kinases in cHL cell lines. Cell lysates of cHL cell lines were probed with antibodies specific for p-Akt(Thr308), p-Akt(Ser473), and p-ERK. The antibody to ACTB and to the total AKT and ERK were used as loading controls. (B) AKT and ERK inhibitors restore FOXO1 expression in cHL cell lines. A total of 1 × 10⁶ cells were seeded in 3 mL of complete medium per well of a 6-well plate. The KP372-1 or U0126 kinase inhibitors were added at the same day. Twenty-four hours later, the cells were harvested and FOXO1 expression was detected by immunoblot. (C) Sensitivity of the cHL cell line to AKT inhibitor KP372-1 and to ERK inhibitor U0126. A total of 2 × 10⁴ cells were seeded per well of a 96-well plate simultaneously with the kinase inhibitors at different concentrations. Sixty hours later, the viability of the cells was assessed by MTT staining. IC₅₀ of U0126 and KP372-1 for p-ERK or p-AKT negative (−) and positive (+) cHL cell lines, respectively. The experiments were done in triplicate.