Knockdown of C/EBPa blocks the differentiation induced by FLT3 inhibition. (A) Molm14 cells were cocultured with stroma in the presence of increasing concentrations of quizartinib for 24 hours, and then cells were collected and analyzed for morphology. For each condition, 100 viable cells were counted and categorized as undifferentiated or differentiated. (B) Molm14 cells (2 million per sample) were incubated with siRNA for C/EBP-α (or scrambled control), electroporated, and then cocultured with stroma. After 24 hours, the cells were harvested, lysed, and analyzed for C/EBPα protein levels by Western blotting. Incubation for 48 hours led to identical results (not shown). (C) Molm14 cells were subjected to siRNA C/EBPa knockdown as in panel B, cocultured with stroma for 24 hours, and then treated with 5nM quizartinib. After 24 hours, the cells were harvested and examined and scored for morphologic changes as in panel A.