Effect of SF on the levels of total and phosphorylated proteins involved in the G2/M cell cycle arrest.

<p>(A) The protein levels of KLF4, p21, p53, cyclin B1, cyclin D1, cdc2, and cdc25C and phosphorylation of cdc2 (Tyr 15) and cdc25C (Ser 216) were evaluated by immunoblots in LCL, pre-B ALL, and T-ALL cell lines incubated with 7.5 µM SF or vehicle for 24 hours. β-actin was used as a loading control. The data are representative of three independent experiments. (B) Detection of phospho-H2AX (pH2AX) in nuclei by flow cytometry in Nalm-6 and Jurkat cells treated with 7.5 µM of SF or Etoposide (ETO) for 24 hours. The data are representative of three independent experiments. (C) Diagram depicting the role of SF in the G2/M cell cycle arrest of ALL cells.</p

<i>In vivo</i> anti-leukemic effect of SF in ALL xenograft models.

<p>(A) Nalm-6 cells were transduced with the FFluc retrovirus and injected intravenously into NOD/SCID mice. Twenty-four hours later, the mice were treated with SF or vehicle (2 mg i.p. daily) and monitored weekly for the distribution of leukemic cells by bioluminescence imaging (BLI). (B) The Nalm-6-FFluc cells were mixed with a high-protein matrigel and injected into the flank of NOD/SCID mice. One week later, tumor establishment was confirmed by BLI; then, the mice were treated by oral gavage (2 mg/gave, twice daily) for 7 days. The total counts were determined for the control- and SF-treated mice. The data are representative of three independent experiments. The statistical significance was calculated using the two-tailed Student’s <i>t</i>-test.</p

SF induces apoptosis selectively in ALL cell lines.

<p>(A) Apoptosis was evaluated by annexin-V and 7-AAD staining of LCL, Nalm-6, Jurkat and KOPTK1 cells cultured in the absence or presence of 7.5 µM SF. The data are representative of three independent experiments. (B) The percentages of annexin-V-positive cells were determined for each cell line cultured in the presence or absence of SF. (C) SF activates the proteolytic cascade of caspases and PARP in leukemic cells. LCL cells (control), pre-B ALL cells (Nalm-6, REH, and RS-4), and T-ALL cells (Jurkat, RPMI, DND41, and KOPTK1) were incubated with 7.5 µM SF for 24 hours and analyzed by immunoblotting. The arrows indicate the cleaved forms of the caspases and PARP. β-actin was used as a loading control. The data represent the mean and standard deviation (n = 3). *** <i>P</i><0.001 (two-tailed Student’s <i>t</i>-test).</p

SF inhibits the AKT/mTOR survival pathway in leukemic cells.

<p>The protein levels of phosphorylated and total AKT and mTOR were examined by immunoblotting. LCL, pre-B ALL, and T-ALL cell lines were incubated with 7.5 µM SF or vehicle for 24 hours. β-actin was used as a loading control. The data are representative of three independent experiments.</p