Effect of various treatments on cytarabine sensitivity in HeLa cells. (A) Cells were treated with diluent or the indicated concentrations of cytarabine (Ara-C) for 4 hours. After cells were washed, postnuclear lysates were prepared. Equal amounts of protein were electrophoresed and sequentially immunoblotted for phospho-Ser345-Chk1 (P-Chk1) and total Chk1. (B) Forty-eight hours after the second transfection with control or Chk1 siRNA, cells were harvested for immunoblotting with anti-Chk1 and antiactin antibodies (inset) or treated for 24 hours with the indicated concentration of cytarabine. At the completion of the drug incubation, colonies were allowed to form under drug-free conditions. (C) Forty-eight hours after the second transfection with control or PDK1 siRNA, cells were harvested for immunoblotting with anti-PDK1, anti-Thr308-phospho-Akt (P-Akt), and anti-Akt antibodies (inset) or treated for 24 hours with the indicated concentration of cytarabine. At the completion of the drug incubation, colonies were allowed to form under drug-free conditions. (D) In parallel experiments, cells were treated for 24 hours with diluent or the indicated concentration of cytarabine, and diluent or 20 μM LY294002 was then added for an additional 24 hours in the continued presence of diluent or cytarabine. At the completion of the drug incubation, cells were harvested for immunoblotting with antiphospho-Thr308-Akt, anti-Akt, antiphospho-Ser9/21-GSK3α/β, and anti-GSK3β antibodies (inset) or washed and allowed to form colonies under drug-free conditions. Error bars: mean ± standard deviation of triplicate aliquots. Each panel is representative of 3 or more independent experiments with similar results. HeLa-cell cloning efficiency ranged from 70% to 90%. The total period of cytarabine exposure was 48 hours in panel D but only 24 hours in panels B and C, explaining the enhanced sensitivity in panel D.