Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed

Carole Gauron2#, Christine Rampon1,2#, Mohamed Bouzaffour1,4, Eliane Ipendey2,3, Jérémie Teillon2, Michel Volovitch2,3, and Sophie Vriz1,2,*

Supplementary Information

Figure S1: ROS detection at 24 hpa and after NOX inhibition Figure S2: Time course of wound healing after lesion of the adult caudal fin Figure S3: Spatio-temporal control of cell death during caudal fin regeneration Figure S4: Apoptosome inhibition interfered with progenitor cell marker expression Figure S5: Figure 2 c and d full-length western blot ------Figure S1: ROS detection at 24 hpa and after NOX inhibition ROS are detected with a fluorescent probe (H2DCFDA) (a) High magnification of fins 12-18 hpa shows dynamic production of ROS during this period (b) at 24 hpa isolated cells in the stump epidermis are positive for ROS either in the proximal than in the distal part of the fin (arrows). (c) ROS are detected with H2DCFDA at 6 hpa. Fish were incubated in VAS2870 1 M or vehicle. NOX inhibition reduced ROS detection. R: ray; IR: inter-ray.

1 Figure S2: Time course of wound healing after lesion of the adult caudal fin Caudal fin was cut in between rays and allow to form a wound. Representative images are shown. In these conditions wound closure is complete after 5 hours after lesion (hpl).

Figure S3: Spatio-temporal control of cell death during caudal fin regeneration (a-b) Controls for cell death detection. Negative (without reagent, left panel) and positive (DNase treatment, middle panel) controls were included in the TUNEL assay. After TUNEL assay on the whole fin, positive controls were sectioned (transversal sections) (right panel) to verify that cell death could be detected in mesenchymal cells. R: ray; IR inter-ray. (b) TUNEL assay performed on sections. Fins were cryosectioned (transversal sections) before TUNEL assay. The distribution of positive cells was equivalent to that in the treated whole fin; in particular, positive cells were detected in the epidermis and very little in the mesenchyme. DNase: positive control; 18 hpa: fin allowed to regenerate for 18 hours. Scale bars = 100 m.

Figure S4 : Apoptosome inhibition interfered with progenitor cell marker expression. Gene expression was analyzed by quantitative RT-PCR on the fin allowed to regenerate 18 hpa after NS3694 (5 M) or vehicle (DMSO) treatment. The DMSO-treated sample was taken as 1. Error bars represent the SEM (* p<0.05, **p< 0.01, ***p < 0.001).

2 Figure S5: Figure 2 c-d full-length western blot. See main text for details

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