Adjudin improves the function of regenerated beta cells in zebrafish. (a) The z score of bioluminescence during beta cell regeneration. Tg(ins:NLuc);Tg(ins:Flag-NTR) larvae were treated with DMSO or 5 µmol/l adjudin for 1 day after beta cell ablation, and bioluminescence was analysed at 5 dpf. Mann–Whitney test: ***p<0.001. n=24 larvae per treatment (3 larvae per data point). Data are presented as mean ± SEM. (b) qPCR analysis of ins expression in zebrafish larvae during beta cell regeneration. After beta cell ablation, Tg(ins:Flag-NTR) larvae were treated with DMSO or 5 µmol/l adjudin for 1 day from 4 to 5 dpf before being analysed by qPCR. Mann–Whitney test: *p<0.05. n=80 larvae per treatment (5–12 larvae per data point). Data are presented as mean ± SEM. (c) Free glucose levels in zebrafish larvae during beta cell regeneration. Tg(ins:CFP-NTR) larvae were treated with DMSO or 5 µmol/l adjudin for 2 days after beta cell ablation, and free glucose levels were analysed at 6 dpf. Mann–Whitney test: *p<0.05. n=12 larvae per treatment. Data are presented as mean ± SEM. (d) Maximum projections of primary islets in zebrafish during beta cell regeneration. Tg(ins:H2BGFP);Tg(ins:Flag-NTR) larvae were treated with DMSO or 5 µmol/l adjudin for 2 days after beta cell ablation, and subsequently fixed at 6 dpf for analysis. Scale bar, 10 μm. (e) Quantification of total number of regenerated beta cells in (d). Mann–Whitney test. n=27 larvae (DMSO), n=33 larvae (adjudin). Data are presented as mean ± SEM. (f–i) Representative images from live calcium recording of islets before (f and h) and after the addition of 200 mmol/l glucose to the E3 medium (g and i). Beta cells expressing H2BmCherry are shown in red and the calcium signal in green. Images with merged channels are shown in (f, g, h, i), single red channels are shown in (f’, g’, h’, i’), and single green channels are shown in (f’’, g’’, h’’, I’’). Tg(ins:GCaMP6s);Tg(ins:H2BmCherry);Tg(ins:Flag-NTR) zebrafish larvae were treated with DMSO or 5 µmol/l adjudin for 1 day after beta cell ablation from 3 to 4 dpf, and imaged at 5 dpf. Scale bar, 10 µm. (j) Quantification of relative GCaMP intensity in beta cells at baseline (f and h). Mann–Whitney test: ***p<0.001. n=20 cells from four larvae (DMSO), n=22 cells from four larvae (adjudin). (k) Calcium activity indicated by normalised fluorescence over time in regenerated beta cells. Each line represents one cell. The addition of 200 mmol/l glucose is indicated by the white dashed line. Tg(ins:GCaMP6s);Tg(ins:H2BmCherry);Tg(ins:Flag-NTR) zebrafish larvae were treated with DMSO or 5 µmol/l adjudin for 1 day after beta cell ablation from 3 to 4 dpf, and had calcium signal recorded at 5 dpf. n=6 cells per treatment. (l) Quantification of percentage of beta cells that had calcium response to glucose. Tg(ins:GCaMP6s);Tg(ins:H2BmCherry);Tg(ins:Flag-NTR) zebrafish larvae were treated with DMSO or 5 µmol/l adjudin for 1 day after beta cell ablation from 3 to 4 dpf, and imaged at 5 dpf. Mann–Whitney test: *p<0.05. n=7 larvae per treatment. Data are presented as mean ± SEM. ΔF/F0, signal-to-baseline ratio; ΔF, deviation from the baseline; F0, basal fluorescence
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