Fig. 3.

Trolox reduces the number of apoptotic clusters and oxidative stress in zebrafish embryos injected with 10 pg poly-GR. (A) Upper panel: maximum projection of z-stack images of the SecA5 fluorescent reporter line embryos 48 h after injection with 10 pg poly-GR and treated with 50 µM Trolox or DMSO only. Lower panel: maximum projection of TUNEL staining of the same treatment groups in wild-type AB fish at 2 dpf. (B) Quantification of SecA5 z-stack images (mean±s.e.m.). N=30 fish per group per day. Kruskal–Wallis test (P<0.0001). Dunn's post-hoc multiple comparison test (***P<0.0003). (C) MitoSOX red staining in 2 dpf wild-type AB embryos after injection with 10 pg poly-GR or 400 pg mCherry only, and treated with DMSO only or Trolox (dissolved in DMSO). MitoSOX signal (magenta) is seen as clusters in the brain and in the spinal cord in the tail. MitoSOX staining also shows ROS reactivity in pigmented cells (arrowheads) and along the tail and head (arrows). (D) MitoSOX signal is significantly higher in 10 pg poly-GR compared to mCherry-only-injected zebrafish embryo tails at 2 dpf. N=8 fish per group (mean±s.e.m.). Two-tailed unpaired Student's t-test (P=0.0002). F-test for equal variances (P=0.914) demonstrated that variances were not significantly different. (E) MitoSOX signal is significantly reduced in 10 pg poly-GR-injected zebrafish embryo tails treated with Trolox versus DMSO only at 2 dpf. N=9 fish per group (mean±s.e.m.). F-test (P=0.045) demonstrated that variances were significantly different. Two-tailed unpaired Student's t-test with Welch's correction P=0.01. H₂O₂-treated embryos used for MitoSOX positive control experiments, with an additional n=8 fish per group, are shown in Fig. S4. **P<0.001, ***P<0.0001. Scale bars: 100 µm.