Biological relevance of STAT5 activation in WM. (A) Expression of STAT5A and STAT5B was individually inhibited through the use of a doxycycline-inducible shRNA system transduced into MWCL-1 cells. After a 72-hour treatment with doxycycline (500 ng/mL), cells were lysed and immunoblotted for STAT1, STAT3, STAT5A, STAT5B, and actin, and protein expression was compared with that observed in cells transduced with an empty vector shRNA (EV). Western blotting was performed on 3 separate experiments. Representative blots are shown. (B) After 72-hour induction of STAT5A or STAT5B shRNA with 500 ng/mL doxycycline, IgM was measured by enzyme-linked immunosorbent assay (B), cellular proliferation was measured by ³H-TdR incorporation (C), and levels were compared with those detected in the empty vector-transduced cells (EV). (D) Effect of a small molecule inhibitor of STAT5 on STAT5 activation status in BCWM.1 and MWCL-1 cells. Shaded histograms represent isotype control, dotted lines represent baseline STAT5 phosphorylation, and solid lines represent STAT5 phosphorylation after 24-hour treatment with 10 μM N′-[(4-Oxo-4H-chromen-3-yl)methylene] nicotinohydrazide. Representative plots of 3 separate experiments are shown. (E-H) BCWM.1 and MWCL-1 cells were treated with increasing concentrations of N′-[(4-Oxo-4H-chromen-3-yl)methylene] nicotinohydrazide or IQDMA for 48 hours. IgM was measured by enzyme-linked immunosorbent assay (E,G), and cellular proliferation was measured by ³H-TdR incorporation (F,H). All experiments were performed in triplicate. *P < .05 as determined by the Student t test.