Figure 2

Inhibition of STAT3 reduces colony formation by TKI-resistant CML cells. (a, b) TKI-resistant CML cell lines were retrovirally transduced with shRNA targeting STAT3 (shSTAT3) or scrambled control (shSCR), and cultured in semisolid medium ± imatinib (1.0–2.5 μM). STAT3 and not STAT5 knockdown was confirmed by immunoblot analyses (a, b, right). shSTAT3 reduced colony formation of K562^R (a, left, n=4) and AR230^R (b, left, n=4) cells in the presence of imatinib, with no effect on parental TKI-sensitive controls. (c, d) TKI-resistant CML cell lines were transduced with dominant-negative STAT3 mutants (dnSTAT3) or empty vector (EV) and cultured in semisolid medium ± imatinib (1.0 μM). Inhibition of pSTAT3^Y705 was confirmed by immunoblot analyses (c, d, right). dnSTAT3 reduced colony formation of K562^R (c, left, n=4) and AR230^R (d, left, n=3) cells with no effect on parental TKI-sensitive controls (n=2). (e, f) K562^R (e, n=4) and AR230^R (f, n=4) cells were incubated in methylcellulose semisolid medium with SF-1-066 (1–10 μM) ± imatinib (1.0 μM). SF-1-066 reduced colony formation of only TKI-resistant and not TKI-sensitive cells. (g) Mononuclear cells (MNCs) from peripheral blood of normal donors (n=2) or CML^CD34+ cells from newly diagnosed patients (n=4) were treated ex vivo with SF-1-066 (10 μM) ± imatinib (2.5 μM) in RM or HS-5 CM for 96 h followed by colony-forming assays. All data are represented as percent of controls. Error bars represent s.e.m. *P<0.05; **P<0.01; ***P<0.001.