Fig. S6

AG1 attenuates ROS-induced pericardial edema in a G6PD-dependent manner. a AG1 is not toxic to zebrafish embryos at concentrations <10 μM. Pericardial edema induced by chloroquine treatment was partially attenuated by AG1 treatment. Embryos were treated at 24 hpf with indicated concentrations of AG1 for 24 hours. Severity of pericardial edema is indicated by color schemes provided on the right of the graph. b Ventral views of whole-mount o-dianisidine staining of WT embryos at 48 hpf. Representative staining is shown on the left for embryo scoring; distribution of staining shown on the right. Raw counts used for chi-square analysis and calculated p value are included in table below. Scale bar: 300 μm. c, d Genetic loss of tbx16 function led to mesodermal defects in embryos obtained by incrossing tbx16 mutant heterozygotes. Pre-treatment at 24 hpf with 1 μM AG1 for 24 hours did not prevent pericardial edema that developed at 48 hpf in tbx16 mutants (c) nor was edema rescued at 72 hpf (d), when treated with AG1 at 48 hpf. Phenotypic ratio is indicated at the upper right corner of each image. Embryo orientation is lateral view, anterior left. Scale bar: 500 μm. e Embryos injected with sgRNA (Guide Alone) against exon 10 of g6pd and sgRNA+Cas9 protein (Guide+Cas9, G6PD KO (knockout)) were pooled at indicated times and analyzed for the G6PD protein level by Western blot. f Ventral views of whole-mount o-dianisidine staining of embryos injected with sgRNA (Guide Alone) and sgRNA+Cas9 protein (Guide+Cas9, G6PD KO) at 48 hpf. Scale bar: 300 μm. Representative staining is shown on the left for embryo scoring; distribution of staining shown on the right. Raw counts used for Fisher exact test and calculated exact probability are included in table below. Molecular weight marker is provided on the left of the blots. CQ: chloroquine; WT: wild-type; KO: knockout; hpf: hours post fertilization; Veh: vehicle.