|
Fig. 1 ROS drive wound relaxation. (A) Workflow of zebrafish embryo fin fold amputation. Embryos at 3 dpf were amputated just posterior to the notochord. ROS intensity was quantified at the wound edge. The width of the wound is plotted over time to show wound contraction and relaxation. (B) Relative ROS production at the wound edge of NAC-treated (n = 67), DPI-treated (n = 59), and DMSO control groups (n = 63). Fish were treated for 1 h before wounding and maintained in the inhibitor for 1 h postwounding. ROS production at 20 min postamputation (mpa) was normalized to 0 mpa for each group. Data are presented as mean ± SE from three independent experiments (N = 3). (C) Wound widths at the indicated time points were normalized to 0 mpa for the NAC-treated or DMSO control group (n = 9). Data are presented as median ± interquartile range (IQR), pooled from three independent experiments (N = 3). (D) Representative images of wound dynamics in the DPI-treated and DMSO control group at 0, 15, 30, and 60 mpa. Scale bar, 50 μm. (E) Wound widths at the indicated time points were normalized to 0 mpa for the DPI-treated (n = 9) and DMSO control (n = 10) groups. Data are pooled from three independent experiments (N = 3). (F) Workflow of zebrafish embryo fin fold amputation with duox knockdown. One-cell stage embryos were injected with either duox + p53 or p53 morpholino. Embryos at 3 dpf were wounded as described in (A). In addition, mRNA was collected to validate the duox knockdown. (G) RT-PCR was used to confirm the MO-induced splicing defect (N = 3), and in each independent experiment, n = 8 for each group. (H) Relative ROS production at the wound edge of p53-injected control (n = 48) and duox + p53-injected groups (n = 41). Data are presented as mean ± SE from three independent experiments (N = 3). (I) Representative images of wound dynamics in the duox + p53-injected group and the p53-injected control group at 0, 15, 30, and 60 mpa. Scale bar, 50 μm. (J) Wound widths at the indicated time points were normalized to 0 mpa for duox + p53-injected (n = 9) and p53-injected control (n = 9) groups. Data are presented as median ± IQR, pooled from four independent experiments (N = 4). (B) One-way ANOVA. (C, E, and J) Mann-Whitney test. (H) Unpaired t-test.