Fig. 4

ROS inhibit MRLC phosphorylation and F-actin. (A) Proposed mechanism. ROS inhibit the phosphorylation of MRLC and reduce actin contraction. ROCKOUT inhibits ROCK, ML-7 inhibits MLCK, and Bleb inhibits NMII. (B) Schematic of wound dynamics, closure, and regrowth assay for ROCKOUT-treated, ML-7-treated, and DMSO control groups and immunofluorescence staining assays for DPI-treated and DMSO control groups (C) Representative images of immunofluorescent staining of p-MRLC for DPI-treated and DMSO control groups unwounded (UW) and at 2 and 15 mpa. Embryos were also stained with phalloidin and DAPI to reveal F-actin and the nucleus. Scale bar, 50 μm. (D and E) p-MRLC and F-actin intensity of DPI-treated and DMSO control groups at the indicated time points. are presented as median ± IQR, pooled from two independent experiments (N = 2). (DMSO control UW group at n = 43, DPI UW group at n = 40, DMSO control group at 2 mpa [n = 39], DPI group at 2 mpa [n = 36], DMSO control group at 15 mpa [n = 40], DPI group at 15 mpa [n = 32]) (F) Changes in normalized tailfin width over time in the ROCKOUT-treated group and DMSO control group. are presented as median ± IQR, pooled from three independent experiments (n = 9; N = 3). (G) Regrowth of ROCKOUT-treated and DMSO control at 0, 1, and 2 dpa. Data are presented as median ± IQR, pooled from three independent experiments (n = 60; N = 3). (H) Changes in normalized tailfin width over time of the ML-7-treated and DMSO control groups. Data are presented as median ± IQR, pooled from three independent experiments (n = 9; N = 3). (I) Regrowth of ML-7 and DMSO control group at 0, 1, and 2 dpa. Data are presented as median ± IQR, pooled from three independent experiments (n = 60; N = 3). (J) Changes in normalized tailfin width over time of ML-7-treated (n = 9) and ML-7, DPI double-treated groups (n = 10). Data are presented as median ± IQR, pooled from three independent experiments (N = 3). (K) Representative images of ROCKOUT-treated, ML-7-treated, and DMSO control group’s x-z plane amputated wounds at 0 and 30 mpa. Blue, segmented nuclei. Scale bar, 10 μm. (L) Relative distances of nuclei at the wound edge to the central plane of the wound in ML-7-treated, ROCKOUT-treated, and DMSO control groups at 0 and 30 mpa. Data are presented as median ± IQR, pooled from two independent experiments (DMSO control group at 0 mpa [n = 28], DMSO control group at 30 mpa [n = 35], ML-7 group at 0 mpa [n = 27], ML-7 group at 30 mpa [n = 20], ROCKOUT group at 0 mpa [n = 30], ROCKOUT group at 30 mpa [n = 26; N = 2]). (M) Wound widths at the indicated time points were normalized to 0 mpa for the Bleb-treated or DMSO control group (n = 9). Data are presented as median ± IQR, pooled from three independent experiments (N = 3). (N) Representative images and quantitation of percentages of larvae with dextran penetration in the Bleb-treated and DMSO control group at 3 and 30 mpa (DMSO group at 3 mpa [n = 12], Bleb group at 3 mpa [n = 12], DMSO group at 30 mpa [n = 12], Bleb group at 30 mpa [n = 12]). 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. (F, H, J, and M) Mann-Whitney test. (D, E, G, I, and L) Aligned rank transform.