H-RasV12 induces ROS accumulation in living zebrafish larvae and ROS scavenging or NOX4 inhibition prevents Ras-induced larvae defects and death. (a) Ras-induced ROS can be detected in a living animal. A zebrafish strain carrying heat-inducible eGFP.H-RasV12 was heat-shocked and 27 h post-induction either control or eGFP.H-RASV12 transgenic animals were visualized by live imaging for GFP expression and ROS levels with the DHE probe. Scale bar: 100 µmm. (b) NOX4 inhibition reduces H-RasV12-induced ROS accumulation in zebrafish larvae as detected by live imaging by DHE. Scale bar: 200 µmm. (c) The observed phenotypes following heat shock (HS) in wild type (I) and H-RasV12 transgenics (II–IV) are displayed in each panel:I, normal animal; II, heart defects; III, heart and craniofacial defects; IV, heart, craniofacial and body defects; V, wild-type animal treated with DMSO after HS; VI, H-RasV12 transgenic animal treated with DMSO after HS; VII, the level of GFP-Ras in the larvae depicted in VI; VIII–X, heat-shocked larvae treated with 1–5 µmM of Nox4i; VIII, wild-type animal treated with Nox4i after HS; IX, H-RASV12 transgenic animal treated with Nox4i after HS; X, level of GFP-Ras in the larvae depicted in IX. Scale bar: 100 µmm. (d) The quantification of the occurrence of the phenotypes (demonstrated in c) following different treatments shows that ROS scavenging or Nox4 inhibition rescues the detrimental effects of RasV12 expression. Differences between the percentages of phenotypes observed in H-Rasv12 transgenics treated by NAA versus NAC and DMSO versus Nox4i are statistically significant scored for death and also heart, craniofacial and body defects (*P-value < 0.01)