C188-9 inhibits G-CSF–induced expression of Stat3 target genes and induces apoptosis in AML cell lines. (A) G-CSF–induced expression of SOCS3 and PIM1 mRNA in 3 cell lines is shown. Cells were treated with 10μM C188-9 for 1 hour or left untreated, and then stimulated with 100 ng/mL G-CSF for 1 hour. Fold change in mRNA level was quantified by the ΔΔC_t method as described in “Quantitative RT-PCR.” Values are the mean ± SEM of at least 2 independent experiments. NB4 cells also were treated with C188-9, then stimulated with 1μM ATRA for 1 hour, and the relative levels of the ATRA-inducible gene CCL24 were measured. (B) C188-9 induced apoptosis in AML cell lines. Cells were treated for 24 hours with C188-9; then apoptotic cells were quantified by labeling with annexin V–PE. The baseline percentage of annexin V⁺ cells in an untreated sample was subtracted from each drug-treated sample. Dose-response curves were generated and EC₅₀ values estimated, as shown. Each value represents the mean ± SD for at least 3 replicates.