Effect of Star 27 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. (A–D) The effect of Star 27 on zebrafish embryonic development at 3 dpf, showing no noticeable morphological defects up to 10 μM. (E–I) The effect of PKC412 on zebrafish embryonic development at 3 dpf, showing substantial pericardial defects beginning at 500 nM (data not shown), and tail curvature and length defects beginning at 1 μM and 2.5 μM, respectively vs. vehicle. (J, K, M) The effect of 10 μM Star 27 treatment on mpo+ myeloid cells development in the posterior blood island (PBI) at 30 hpf, showing no statistically significant change. (J, L, M) The effect of 10 μM PKC412 treatment on mpo+ myeloid cells development in the PBI at 30 hpf, showing a statistically significant granulogenesis/myelosuppression. (N–P) Three categories of mpo transcription (N, normal; O, intermediate; P, severe) were defined based on the WISH results (three experiments). (Q) The rescue effect of 10 μM Star 27 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing rescue of normal phenotype approaching that seen for AC220 (He et al., 2014). Scale bar equals 500 μm. Blue arrows indicate the pericardial edema, green arrows indicate tail shortening and curving, and red arrows indicate the FLT3-ITD AML mpo+ myeloid cells expansion. PKC412 treatment on FLT3/ITD-induced mpo+ myeloid cells expansion at 30 hpf, showing an efficaciousness similar to Star 27, consistent with Figures 1–3. For all experiments: Zebrafish embryos were collected and kept in standard E3 medium at 28°C. Different concentrations of either drug were added to the E3 medium from 6 hr post fertilization (hpf) to 3 days post fertilization (dpf). Embryos treated with DMSO or 10 μM drug from 6 to 30 hpf were collected for mpo whole mount in situ hybridization (WISH) analysis. 80 ng plasmid DNA containing FLT3/ITD sequence was microinjected into one-cell stage embryos, and the uninjected embryos were used as control. FLT3/ITD-injected embryos were treated with DMSO or 10 μM drug from 6 to 30 hpf. Embryos were collected at 30 hpf for mpo WISH analysis. **p <0.01.