NSC140873, Ro5-3335, and Ro24-7429 inhibit definitive hematopoiesis in zebrafish embryos. (A) NSC140873 reduced circulating cd41-GFP⁺ (Upper) and lck-GFP⁺ (Lower) cells in transgenic zebrafish embryos compared with DMSO-treated embryos. Red arrows point to circulating cd41-GFP⁺ cells in the heart and lck-GFP⁺ cells in the thymus, respectively. (B) Dose-dependent reduction of circulating cd41-GFP cells by NSC140873. (C) Chemical structures of the three related compounds. (D) Dose-dependent reduction of circulating cd41-GFP cells by Ro5-3335 and Ro24-7429. (E) The runx1 W84× truncation mutation sensitizes fish embryos to the inhibition by Ro5-3335. *Numbers of untreated runx1^+/- embryos or DMSO-treated runx1^+/- embryos with intermediate or no visible circulating CD41-EGFP⁺ cells were compared. ^#Numbers of runx1^+/- embryos or wild-type embryos with intermediate or no visible circulating CD41-EGFP⁺ cells after Ro5-3335 treatments were compared. For all comparisons in B, D, and E, the P values were calculated with 2 × 2 contingency tables and Fisher?s exact test.

Ro5-3335 rescues hematopoietic defect in the RUNX1?ETO transgenic embryos. (A) RUNX1-ETO transgenic embryos at 48 h postfertilization (hpf). HS, heat shock. Arrows point to the posterior blood island. Note the pooling of blood cells in the heat-shocked embryo. (B) Heat-shocked RUNX1-ETO transgenic embryos at 48 hpf that were treated with either 25 μM Ro5-3335 (Upper) or DMSO (Lower). (C) Bar graphs showing the effect of incubating heat-shocked RUNX1-ETO transgenic embryos with Ro5-3335 at the indicated concentrations. Black bars, embryos with blood pooling and no circulation; gray bars, embryos without blood pooling but also no circulation; white bars, embryos without blood pooling and with circulating cells. Means of three independent experiments are shown with SDs. *P < 0.03; **P < 0.003.