Fig. 3

ROS promote wound closure and fin regrowth. (A) Workflow of the zebrafish embryo fin fold wound closure assay. The nuclei of the enveloping layer are labeled with GFP, and their distance to the wound center is measured at 30 mpa. (B) Representative images of the x-z plane of the wound edge of DPI-treated and DMSO control groups at 0 mpa. Scale bar, 10 μm. (C) Representative images of indicated planes of the wound edge of DPI-treated and DMSO groups at 30 mpa. Scale bar, 5 μm. (D) Representative images of the x-z plane of the wound edge of duox + p53-injected and p53-injected control at 0 and 30 mpa. Blue indicates nuclei masks in (B–D). Scale bar, 10 μm. (E and F) Distances of nuclei at the wound edge to the wound center of inhibitor-treated or morpholino-treated groups at 0 and 30 mpa (DMSO control group at 0 mpa (n = 28), DMSO control group at 30 mpa (n = 35), DPI group at 0 mpa (n = 28), DPI group at 30 mpa (n = 33); p53 control group at 0 mpa (n = 19), p53 control group at 30 mpa (n = 54), duox morphants at 0 mpa (n = 57), duox morphants at 30 mpa (n = 33); n stands for the number of nuclei, the number of larvae in each group 3). Data are presented as median ± IQR, pooled from two independent experiments (N = 2). (G) Dextran wound entry assay for the DPI-treated group and DMSO control. Representative images of dextran penetration in the DPI-treated and DMSO control group at 3 and 30 mpa are shown in the left panel. The percentage of larvae with dextran in the wounded tissue of each condition is shown in the right panel (DMSO group at 3 mpa [n = 12], DPI group at 3 mpa [n = 14], DMSO group at 30 mpa [n = 12], DPI group at 30 mpa [n = 12]). (H) Dextran wound entry assay with duox knockdown. Representative images of dextran penetration of duox morpholino-treated and p53 control groups at 3 and 30 mpa are shown in the left panel. The percentage of larvae with dextran in the wounded tissue of each condition is shown in the right panel (p53 group at 3 mpa [n = 12], duox+p53 group at 3 mpa [n = 12], p53 group at 30 mpa [n = 12], duox + p53 group at 30 mpa [n = 16]). (G and H) Data are pooled from three independent experiments (N = 3). Fisher’s exact test. “+” indicates dextran penetration at the wound, while “–” indicates no entry of dextran at the amputated wound. (I) Workflow of zebrafish embryo fin fold regrowth assay. The distances between the notochord and the fin tip were measured at 0, 1, and 2 dpa. (J) Regrowth of DPI-treated and DMSO control groups at 0, 1, and 2 dpa. Data are presented as median ± IQR, pooled from three independent experiments (n = 60; N = 3). (K) Regrowth of duox morpholino-treated (n = 23) and p53 control (n = 26) groups at 0, 1, and 2 dpa. Data are presented as median ± IQR, pooled from three independent experiments (N = 3). (L) Kaplan-Meier survival chart of morpholino-treated groups postamputation. Data are pooled from three independent experiments (duox+p53 [n = 23], p53 [n = 26; N = 3]). (E, F, J, and K) Aligned rank transform. (L) Log-rank analysis.