A novel Stat3 SH2 small-molecule probe, C188-9, inhibits Stat3 phosphorylation in AML cells. (A) C188-9 inhibited G-CSF–induced pStat3 in a dose-dependent manner, as demonstrated by FACS. Representative histogram overlays for Kasumi-1 and THP-1 cell lines are shown. Light gray area represents unstimulated; dark gray area, G-CSF (100 ng/mL) only; solid line, G-CSF + 3μM C188-9; and dotted line, G-CSF + 10μM C188-9. (B) The dose-response relationship for C188-9 inhibition of G-CSF–induced pStat3 for 4 AML cell lines is shown. The IC₅₀ values for all 6 cell lines were between 4 and 8μM. Each point represents the mean of at least 3 separate experiments. (C) C188-9 inhibited G-CSF–induced pStat3 in a dose-dependent manner but did not alter total Stat3 protein levels, as demonstrated by Western blotting. For both FACS and Western blotting, cells were treated with C188-9 at the indicated dose for 1 hour and then stimulated with G-CSF (100 ng/mL) for 15 minutes. (D) C188-9 did not affect the tyrosine phosphorylation levels of most tyrosine kinases represented on a tyrosine kinase protein array. Duplicate array spots from cells stimulated with 100 ng/mL G-CSF (left) and cells pretreated with 10μM C188-9 before G-CSF (middle) are shown. The ratio of inhibitor/no inhibitor densitometric values is shown in the right-most column. (E) C188-9 did not inhibit the activity of the MAPK/ERK pathway or the Akt pathway, as assessed by pERK1/2 and pAkt immunoreactivities.