Fig. 5.

The HSPC ROS level is elevated in alas2 and alad mutants. (A) Schematic of biochemical assay of oxidative stress biomarkers (ROS, CAT, SOD, GSH and MDA) in zebrafish dissected trunk regions at 36 hpf. (B) Confocal imaging showing the co-localization of kdrl:GFP⁺ and MitoSOX⁺ cells in the VDA of control, alas2^−/− and alad^−/− at 36 hpf. The dorsal aorta (DA) regions are denoted by white dashed lines, and the kdrl⁺/MitoSOX⁺ cells are denoted by white arrowheads. (C) Quantification of kdrl⁺/MitoSOX⁺ cells in B. n=3 experimental replicates. (D) Quantification of mean fluorescence intensity (MFI) of mitochondrial ROS level in HSPCs of control, alas2^−/− and alad^−/− measured by MitoSOX staining. n=3 experimental replicates. (E) Expression of the HSPC marker runx1 in control, alas2^−/− and alad^−/− with or without NAC treatment at 36 hpf examined by WISH. The AGM regions for marker gene-positive cell counting are denoted by red arrowheads. (F) Quantification of the runx1-positive HSPCs in E. n=3 experimental replicates. (G) Expression of the HSPC marker runx1 in control, alas2^−/− and alad^−/− with or without mitoTempo treatment at 36 hpf examined by WISH. The AGM regions for marker gene-positive cell counting are denoted by red arrowheads. (H) Quantification of the runx1-positive HSPCs in G. n=3 experimental replicates. (I) Confocal imaging shows the kdrl⁺/cmyb⁺ HSPCs in control, alas2^−/− and alad^−/− with or without mitoTempo treatment at 36 hpf. (J) Quantification of the HSPCs in I.; n=3 experimental replicates. Number of samples are indicated. Data are mean±s.d. ***P<0.001 (one-way ANOVA, Tukey's multiple comparisons in C,D,F,H,J). n.s., not significant. Scale bars: 100 μm.