Fig. 5

Experimental validation on Zebrafish embryos of drugs predicted to modulate the activity of miR-451 or the expression of its targets.a Depiction of our experimental design. Zebrafish embryos were incubated with different drug candidates predicted by sChemNET in combination with phenyl-thiourea (PTU), a chemical known to induce anemia due to oxidative stress when miR-451 activity is impaired, but not in wild-type embryos. 48 hours after fertilization, embryos display robust blood circulation. At this stage, the accumulation of mature erythrocytes can be easily assessed in transparent embryos using O-dianisidine, a hemoglobin-specific stain. Drugs impairing miR-451 activity induce anemia, while miR-451 boosting drugs will increase erythrocyte production (blood circulation). b Ventral images of 2-day-old embryos stained with O-dianisidine to reveal hemoglobinized cells (brown staining) for wild-type embryos and those treated with docetaxel, -elemene and -calcidol. Blood accumulates in the ventral region (ducts of Cuvier). c Lateral view of another group of embryos to reveal accumulation of the excess of blood in the tail region upon treatment with docetaxel. d Northern blot quantification of miRNA expression in zebrafish embryos treated with drug candidates. Total RNA extracted from 2-day-old embryos was analyzed by Northern blot to reveal the expression of miR-451, miR-144, miR-15, and let-7 under different drug treatments. e Quantification of miRNA expression based on the radioactive signal of miRNA probes after Northern blot assay. Bars represent averaged normalized miRNA expression from three representative experiments (white circles). Error bars indicate the standard error of the mean. **p-value = 0.0091 One-way ANOVA test.