Fig. 5

Mido(L)-ATRA suppresses AKT activity to dephosphorylate RAF S259 and induce cell differentiation. (A) Western blot analysis of phosphorylated Akt in the three cell lines. Changes in phosphorylation were detected at different time points, and the corresponding expression of GAPDH at each time point was used as the internal control. The membranes were cut prior to hybridization and the original blots are presented in Supplementary Fig. 3. The values shown below each lane indicate relative units, with the values in DMSO-treated cells defined as 1.0. The phosphorylated protein/unphosphorylated protein ratios are also shown. HL-60, HL-60Res and U937 cells were pretreated with 2, 2 and 0.5 μM LY294002 (LY) respectively, for 2 h. Then, HL-60 cells were treated with 0.25 μM midostaurin and 0.1 μM ATRA (M (L) + RA) for 3 d. U937 and HL-60Res cells were treated with 0.1 μM midostaurin and 1 μM ATRA (M (L) + RA) for 6 d. The effects of LY294002 on mido(L)-ATRA-induced differentiation were assessed by flow-cytometric analysis of CD11b expression (B and C) and the morphology (D, magnification is 1,000 × .) in the three cell lines. B shows the column graph of flow cytometric data of CD11b expression. ###P < 0.005 versus M(L) + RA-treated cells. Each value indicates the mean ± SD of three independent measurements. (C) The histograms of flow cytometric data of CD11b expression. The results are representative of three independent experiments. (E) HL-60, HL-60Res and U937 cells were pretreated with LY294002 (LY), and were then treated with M(L) and ATRA as mentioned above for 0.5 and 2 h. Phosphorylation of Akt and RAF S259 was analyzed by Western blotting in the three cell lines. GAPDH was used as the internal control. The membranes were cut prior to hybridization and the original blots are presented in Supplementary Fig. 3. The values shown below each lane indicate relative units, with the values in DMSO-treated cells defined as 1.0. The phosphorylated protein/unphosphorylated protein ratios are also shown