Silencing WT1 expression decreases colony formation capacity and enhances the sensitivity of leukemia cells to etoposide and 17-AAG. (A) K562 cells were transiently transfected with nonspecific (NS) and WT1 shRNA plasmids. After 48 hours, silencing of WT1 protein and the downstream WT1-regulated proteins Bcl-2 and c-Myc were analyzed by Western blotting. (B) K562 cells stably expressing NS or WT1 shRNA were plated in triplicate in semisolid media, and colony formation was scored after 10 days. Colonies were defined as aggregates of greater than 50 cells. Data represent means ±SDs of triplicates (**P < .005). (C) K562 cells stably expressing NS or WT1 shRNA as in panel B were left untreated or treated with 10μM etoposide for 24 hours. Apoptosis was assessed by annexin V staining and flow cytometry. Data represent means ± SDs of 2 independent experiments (*P < .05). (D) K562 cells were treated with either STA-9090 or etoposide alone or with a combination of both for 72 hours. Viability was assessed by alamarBlue assay and fluorometry. (E) K562 cells stably transfected with NS or WT1 shRNA were left untreated or treated with 5μM 17-AAG for 48 hours. Apoptosis was measured as described in panel C. Data represent means of ± SDs of triplicates (*P < .05).