Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC₅₀ determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate.