Figure 5

Figure 5

ROS concentration is elevated in nnt mutants exposed to ethanol. (A–H) CellROX Assay-stained 24 hpf fish that were unexposed, exposed to 1% EtOH from 6–24 hpf, exposed to NAC from 6–24 hpf, or exposed to 1% EtOH +1 mM NAC from 6–24 hpf. All images are dorsal views of the head with anterior to the left. (A) Unexposed wildtype embryos have lower basal levels of ROS than (E) unexposed mutants (p = 0.0175). (B) 1% EtOH exposed wildtypes have lower ROS levels than (F) EtOH-exposed mutants (p = 0.0022). (C) 1 mM NAC treated wildtypes were not significantly different from (G) NAC treated mutants. (D) Wildtypes treated with 1 mM NAC and 1% EtOH were not significantly different from the (H) NAC and EtOH-treated mutant, but these mutants had significantly elevated ROS compared to the wildtype control (p = 0.0016). (I) Graph depicting ROS concentration across all groups in 24 hpf zebrafish (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (J–Q) CellROX Assay-stained 48 hpf fish. Dorsal views, anterior to the left. (J) Unexposed wildtypes were not significantly different in ROS concentration than (N) unexposed mutants. (K) 1% EtOH-exposed wildtypes were not significantly different from (O) EtOH-exposed mutants. (L) Wildtypes treated with 1 mM NAC were not significantly different from (P) NAC-treated mutants. (M) Wildtypes treated with 1 mM NAC and 1% EtOH were not significantly different from the (Q) NAC and EtOH-treated mutant, though these mutants had significantly lower ROS concentration compared to the EtOH-treated mutants (p = 0.0337). (R) Graph depicting ROS concentration across all groups in 48 hpf zebrafish (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Ctrl: control, EtOH: ethanol, NAC: N-acetyl Cysteine, Mut: mutant, Wt: wildtype.