Fig. 3

Mido(L)-ATRA induces differentiation via MEK-ERK-mediated modulation of the protein levels of C/EBPs and PU.1. HL-60, HL-60Res and U937 cells were pretreated with 1, 5 and 5 μM U0126 (U), respectively, for 2 h. Then, HL-60 cells were treated with 0.25 μM midostaurin (M(L)) and/or 0.1 μM ATRA (RA) for 4 d. U937 and HL-60Res cells were treated with 0.1 μM midostaurin (M(L)) and/or 1 μM ATRA (RA) for 4 and 8 d, respectively. (A) The effect of U0126 on the morphology of HL-60, U937 and HL-60Res cells treated with low-dose midostaurin and ATRA. The magnification is 1,000 × . The results are representative of three independent experiments. Differentiation was also assessed by flow cytometric analysis of CD11b expression in the three cell lines. (B) The column graph of flow-cytometric analysis of CD11b expression in the three cell lines. Each value represents the mean ± SD of three independent measurements. ### P < 0.001 compared with mido(L) + RA. (C) The histograms of flow cytometric analysis of CD11b expression in the three cell lines with the indicated treatment. U937 cells not labeled with the anti-CD11b antibody were used as negative controls, as shown in blue, while U937 cells labeled with the anti-CD11b antibody are shown in pink. The content of CD11b.⁺ cells was deducted from the autofluorescence contribution. The results are representative of three independent experiments. (D) Western blot analysis of phosphorylated ERK1/2, C/EBPβ, PU.1 and C/EBPε in the three cell lines with the indicated treatments for 16 h. The expression of 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 represent relative units, with the values in DMSO-treated cells defined as 1.0. The phosphorylated ERK/ERK ratios are also shown