C188-9 inhibits G-CSF–induced pStat3, induces apoptosis, and reduces colony formation in primary pediatric AML samples. (A) C188-9 inhibited pStat3 induced by treatment with 100 ng/mL G-CSF, as shown by FACS histogram overlays. Shaded area represents G-CSF only; and solid line, G-CSF + 10μM C188-9. (B) The percentage of the uninhibited response was determined from the MFI of the pStat3⁺ population, and dose-response curves were generated. (C) C188-9 induced apoptosis in CD34⁺ primary AML cells. Cells were incubated with C188-9 for 48 hours, and apoptosis was quantified by annexin V staining. (D) C188-9 reduced colony formation in primary AML samples grown in methylcellulose medium with growth factors. The number of colonies (≥ 30 cells; normalized to the number of colonies in untreated control dishes) in each dose of C188-9 is shown for 4 primary samples and 2 normal bone marrow samples. Sample 2187 was first plated at 1 × 10⁵ cells/dish. After 10 days, the number of colonies in the untreated condition was too numerous to count. The untreated cells were harvested and replated in methylcellulose with increasing doses of C188-9, at 1000 cells/dish. CFUs were counted at 14 days. (E) Stat3 activation in AML cells after culture in methylcellulose with growth factors was reduced by the continuous presence of C188-9. pStat3 histogram overlays for CFUs from sample 2187 are shown. Shaded area represents without C188-9; solid line, 1μM C188-9; and dotted line, 10μM C188-9.