Pharmacologic inhibition of ROS formation inhibits FLT3 ITD–mediated cell transformation in a DEP-1–dependent manner. (A) 32D cells, stably transduced with FLT3 ITD, and additionally nontargeting shRNA (control), or DEP-1 targeting shRNA (as indicated), were subjected to colony formation assays in methylcellulose in absence or presence of 1μM DPI (as indicated) for 6 days. Left panel: example images. Pictures were taken with a 4× magnification objective lens using an Olympus CKX41 microscope equipped with a CAMEDIA C-7070 camera (Olympus). Right panel: quantification of colony formation. Only colonies > 0.4 mm were counted. Knockdown efficiencies were assessed by qRT-PCR and by immunoblotting after the treatment period, and are depicted under the graph. Note also equal DEP-1 expression levels in the cells harboring control shRNA. (B) The cells were treated with DPI or were mock-treated in suspension culture for 6 hours, and subsequently lysed and subjected to immunoblotting for assessing STAT5 (left panel) and FLT3 ITD (right panel) activation (representative experiment of 3 with consistent results). (C) Same experiment as in panel A, except that treatment was performed in absence or presence of 5mM TROLOX (TX).