Cell proliferation assay

Regenerates treated with the SU5402 inhibitor were tested for changes in cell proliferation. The cell proliferation assay was carried out using the Click-iT^® EdU HCS Assay (Invitrogen) as described previously (Czarkwiani et al., 2016) and then imaged using confocal microscopy. For each regenerate between ∼100±10 slices were taken per z-stack (1 μm thickness). DAPI-labelled nuclei and EdU-labelled nuclei per stack were counted automatically using the Fiji plugin TrackMate (Tinevez et al., 2016). The number of EdU-labelled nuclei per total number of nuclei ranged from 672/3375 to 1385/5205. The proportion of nuclei labelled with EdU compared with all nuclei labelled with DAPI was calculated as a percentage. Two-tailed unpaired Student's t-test was used and showed no significant difference between control (DMSO) and SU5402-treated samples (t-value=0.261; P>0.25).