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Fig. 3
Withdrawal of DFMO treatment causes partial recovery, and spermidine supplementation rescues phenotypic defects.
(a) Strategy for DFMO treatment and withdrawal. (b) Representative images of control and DFMO-treated Tg(ptf1a:gfp) embryos after 0, 1, or 2 days of DFMO treatment withdrawal; white dotted box denotes the location of the pancreas (c) Quantification of exocrine pancreas length in control (n = 5) and DFMO-treated (n = 5) Tg(ptf1a:gfp) embryos at 3 dpf (p = 0.001); control (n = 6) and DFMO-treated (n = 4) Tg(ptf1a:gfp) embryos at 4 dpf (one day after DFMO was removed)(p = 0.0064); control (n = 7) and DFMO-treated (n = 6) Tg(ptf1a:gfp) embryos at 5 dpf (two days after DFMO was removed)(p = 0.0381); a significant increase in exocrine pancreas length was observed between embryos that were DFMO-treated compared with DFMO + 2 day withdrawal (p = 0.0226). (d) The insulin + β cell population was also quantified for 3 dpf control (n = 5) and DFMO-treated embryos (n = 7) (p < 0.05), as well as 5 dpf control (n = 8) and DFMO + 2 day withdrawal embryos (n = 6) (p < 0.05); no significant difference in β cell number was observed after 2 days of DFMO withdrawal (p = 0.1512). (e) Representative images of ptf1a:GFP + exocrine pancreas cells from control and DFMO + 2 day withdrawal (5 dpf) Tg(ptf1a:gfp) embryos, illustrating the presence of zymogen granules in the ptf1a:GFP + cells. (f) Quantification of exocrine pancreas length in control (n = 12), DFMO-treated (n = 15), spermidine-treated (n = 14), and DFMO + spermidine-treated (n = 20) Tg(ptf1a:gfp) embryos at 72 hpf. Whereas DFMO treatment resulted in reduced exocrine pancreas length (p < 0.0001), the exocrine pancreas in embryos treated with spermidine alone was longer than DFMO-treated (p < 0.0001) but no different from wildtype (p = 0.1843). DFMO-treatment supplemented with spermidine showed exocrine pancreas length no different from wildtype (p = 0.8176) and significantly longer compared with DFMO-treated embryos (p < 0.0001). dpf, days post fertilization; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.