STAT3 phosphorylations regulate regeneration in vitro and in vivo. A Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 6 hours of treatment with vehicle and 1 ng/ml IL-6. B Representative pictures, measurements of scratched area and levels of CXCL1 in supernatants of L929 cells after 24 hours of treatment with vehicle, 5 μM AZD1480 or 12.5 μM PD98059. C Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 0.5 ng/ml IL-6. D Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 200 nM LIF.E Representative pictures and quantification of regenerated area of stat3^+/+,stat3^+/- and stat3^-/- tail fins cut at 3-dpf and analysed at 3 dpa. F Representative pictures and quantification of regenerated area of wild type zebrafish fins cut at 3-dpf and treated for 3 days either with DMSO, 50 μM AG490 or 12.5 μM PD98059. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Generation of stat3^S→A751 zebrafish knock-in line. A Sequence of the donor DNA used to generate the KI. The mutation (gca) is highlighted in red and is flanked by a 36-nt and a 90-nt homology arms. B Representative picture of allele-specific PCRs routinely performed to genotype KI fish: heterozygotes are positive for both alleles, whereas homozygotes are positive for only one. C Morphological analysis (in blue: eye diameter; in orange: eye area; in yellow: body length) and representative pictures of 6-dpf stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 larvae. Scale bar: 200 μm. D Standard light/dark behavioral assay performed on 6-dpf stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 larvae. E Genotype distribution of stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 zebrafish at 6 dpf, 15 dpf, and 60 dpf. F Fluorescent image of Tg(7xStat3-Hsv.Ul23:EGFP)^ia28 intestines of 6-dpf stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 larvae and relative fluorescence quantification. Scale bar: 100 μm. G Expression levels of stat3, socs3a, vegfa and cebpb in 6-dpf stat3^S751/S751 and stat3^A751/A751 larvae. H Regeneration rate of 6-dpf stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 larvae at 3 dpa. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

pS727 Stat3 regulates inflammation, bone metabolism and vitamin D pathway. Representative pictures of regenerated tail fin of stat3^+/+, stat3^+/- and stat3^-/- adult zebrafish and relative quantification. The adult homozygous mutants are rare escapers that manage to reach this developmental stage. B Representative pictures of regenerated tail fin of stat3^S751/S751, stat3^S751/A751 and stat3^A751/A751 adult zebrafish and relative quantification. The adult homozygous mutants are rare escapers that manage to reach this developmental stage. C Expression level of ucmaa, cyp26b1, sp7, mpx, il21, il4, cyp27b1, cyp24a1, vdrb, vdra, and rarga in stat3^+/+ and stat3^-/- larvae. D Expression level of ucmaa, cyp26b1, sp7, mpx, il21, il4, cyp27b1, cyp24a1, vdrb, vdra, and rarga in 6-dpf stat3^S751/S751 and stat3^A751/A751 larvae. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Vitamin D treatment improves tail fin regeneration. A Representative pictures and quantification of regenerated area of wild type zebrafish tail fins cut at 3-dpf and treated for 2 days either with vehicle or 0.5 nM vitamin D. B Expression levels of cyp27b1, cyp24a1, vdrb, vdra and rarga in whole body, tail and regenerated tail samples from 5-dpf larvae treated for 2 days either with vehicle or 0.5 nM vitamin D. C Expression levels of mpx, il4, and il21 in whole body, tail and regenerated tail samples from 5-dpf larvae treated for 2 days either with vehicle or 0.5 nM vitamin D. D Expression levels of cyp26b1, ucmaa, and sp7 in whole body, tail and regenerated tail samples from 5-dpf larvae treated for 2 days either with vehicle or 0.5 nM vitamin D. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Vitamin D partially recovers the regeneration impairment of stat3 mutants. A Representative pictures and relative quantification of stat3^+/+ and stat3^-/- larval tail fins cut at 3 dpf and treated for 2 days with vehicle or 0.5 nM vitamin D. B Representative pictures and relative quantification of stat3^S751/S751 and stat3^A751/A751 larval tail fins cut at 3 dpf and treated for 2 days with vehicle or 0.5 nM vitamin D. Mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

STAT3-VDR molecular interplay. A Analysis of sequences of proximal promoter of vdra, vdrb, cyp24a1, cyp27b1 (A) and stat3 (A'); representative schemes of SBE (A) and VRE (A') consensus sequences. B Expression levels of stat3, socs3a, cebpb and vegfa in wild type 5-dpf zebrafish larvae treated for 2 days with vehicle or 0.5 nM vitamin D. C Expression level of Stat3 and Socs3 in L929 cells treated either with vehicle or 200 nM vitamin D for 24 hours. D Protein level quantification of pSTAT3(S727), pSTAT3(Y705) and pERK1/2, (respectively normalized on STAT3, STAT3 and ERK1/2,) of L929 cells treated either with vehicle or 200 nM vitamin D for 24 hours. Mean ± SEM. *p < 0.05, ****p < 0.0001.