Fig. 4

Fig. 4

AG1 attenuates ROS-induced pericardial edema in a G6PD-dependent manner. a Embryos were treated at 24 hpf with 1 μM AG1 with and without chloroquine (CQ; 100 μg mL⁻¹) and scored at 32 hpf. Representative phenotypic images of pericardial edema and pooling (magenta arrows) are provided on the left (scale bar: 300 μm). Embryo orientation is lateral view, anterior left. Raw counts used for chi-square analysis and calculated p value are included in table below. b ROS levels in individual WT embryos from three independent clutches. Embryos were treated at 24 hpf for 5 h before ROS measurement. Error bars represent mean ± SD (^***p < 0.001, ns = not statistically significant, p > 0.99, Kruskal–Wallis multiple comparison test, adjusted p value using Dunn’s test). c G6PD activity and NADPH levels were measured using the lysates of pooled embryos (from two independent clutches). Error bars represent mean ± SEM (^***p = 0.0003, two-tailed unpaired Student’s t-test). d Embryos were injected with either sgRNA targeting exon 10 of g6pd (Guide alone) or sgRNA + Cas9 protein (Guide + Cas9, G6PD KO (knockout)) to generate G6PD F0 crispants. Representative phenotypic images of pericardial edema and pooling (magenta arrows) are provided on the left (scale bar: 300 μm). Treatment conditions are the same as in a. Raw counts used for chi-square analysis and calculated p value are included in table below. e ROS levels in individual embryos with sgRNA or sgRNA + Cas9 (G6PD KO) protein injection. Treatment conditions and the statistics are the same as in b (^*p = 0.0267, ^**p < 0.01, ^****p < 0.0001, ns = not statistically significant). f G6PD activity and NADPH levels were measured with lysates of pooled embryos from three independent experiments. Error bars represent mean ± SEM of the replicate measurements (^*p < 0.05, ^**p < 0.01, one-way ANOVA for G6PD activity measurement and two-tailed unpaired Student’s t-test for NADPH measurement). Veh: vehicle, KO: knockout, CQ: chloroquine