Figure 4

Calcineurin (CaN) repression potentializes the effect of Notch inhibition on beta cell formation. (A) Experimental design for Notch inhibition test in non-ablated condition. Larvae were treated concomitantly with LY411575 (Notch inhibitor) and Cyclosporin A (CsA) from 3 to 4 dpf and were fixed and analyzed at 6 dpf. (B) Graph representing the mean number of mCherry+ beta cells in the pancreatic tail of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) larvae at 6 dpf depending the concentration of LY411575. The blue dots represent LY411575; and purple combination of LY411575 and CsA. Data are presented as mean values ± SEM. Two-way ANOVA test with Sidak?s multiple comparison test, *p-value <0.05, **p-value <0.05, ns = non-significant. The experiment was performed at least two times and the data are combined in the graph. (C) Whole mount fluorescent immunohistochemistry (GFP and mCherry) of the pancreas of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) larvae at 6 dpf. 3D projection (stack) of one control (without any treatment); one CsA-treated; one LY411757-treated and one with both CsA- and LY411575-treated larvae. The principal islet (PI) and the pancreatic tail are showed. Arrows point out mCherry+ beta cells in the pancreatic tail. Scale 50 µM. (D) Barplot representing the mean number of mCherry+ beta cells in the pancreatic tail of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) larvae at 6 dpf. The black dots represent the control (n=12); gray CsA treatment (n=12);blue LY411575 (n=12); and purple combination of LY411575 and CsA (n=12). Data are presented as mean values ± standard deviation (SD). Two-way ANOVA with Tukey multiple comparison test, ***p-value <0.0005, ****p-value <0.00005. The experiment was performed at least two times and the data are combined in the graph. (E) Graph representing the mean number of mCherry+ beta cells in the pancreatic tail of Tg(ins:NTR-P2A-mCherry); Tg(nkx6.1:GFP) larvae at 5?6?7 dpf. The black dots represent the control; gray CsA treatment;blue LY411575; and purple combination of LY411575 and CsA. Data are presented as mean values ± SEM. Two-way ANOVA test with Sidak?s multiple comparison test, *p-value <0.05. The experiment was performed at least two times and the data are combined in the graph.