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Fig. 5
ROS activity immediately after tail excision. a Time course showing production of ROS after tail excision is reduced in larvae treated with 150??M DPI from 1?h prior to excision. Larvae were bathed in 10??M PFBS-F (205429, Santa Cruz) to detect ROS. b Optimisation to show that treatment for as little as 2?h (1?h pretreatment and 1?h post-treatment) is sufficient to reduce tail regrowth by >50%. Regrowth was quantified from images using the Measure macro in ImageJ by placing a rectangle parallel to the body that started at the end of the notochord and finished at the caudal end of the fin fold. Significance was calculated using one-way ANOVA with Dunnett?s multiple comparisons test and each sample compared to the DMSO control (DMSO n?=?5; 100??M DPI/2?h n?=?4 and P?=?0.0096; 100??M DPI/4?h n?=?5 and P?=?0.0018; 100??M DPI/6?h n?=?3, P?=?0.0015; 150??M DPI/2?h n?=?5, P?=?0.0001; number of experiments?=?1). c Representative larval tails showing the extent of tail regrowth after DPI treatment. d Quantification of PFBS-F fluorescence shows that DPI treatment has a stronger effect on ROS levels than MCI186. Thirty larvae were analysed for each sample except for DPI which had only 29 (number of experiments?=?2). **** indicates P?<?0.0001. e Comparison of the efficacy of DPI to MCI186 in regards to regenerated tail length. Measurements were made as in panel a (DMSO n?=?33, DPI n?=?29, number of experiments?=?3) (DMSO n?=?16, MCI186 n?=?33; number of experiments?=?3). **** indicates P?<?0.0001, *** indicates P?=?0.005. f DPI treatment inhibits wound-induced activation of the Hedgehog, Wnt?-Catenin, RA and FGF pathways. Larvae were pretreated with 150??M DPI for 1?h and for 6 hpa in 100??M DPI. Larvae were then washed in E3 buffer and incubated until 24 hpa or 48 hpf in the case of tcf7 (ptch1 19/19, tcf7 7/9, raldh2 13/19, pea3 9/9). Arrowheads point to expression of pea3 and ptch1 that is found in unoperated animals (see also Supplementary Figure 2)