Fig. S2

Fig. S2

Exogenous administration of the endogenously produced lipid peroxidation product 9-HSA does not trigger apoptosis in the retina and promotes proliferation from 1 dpf in the zebrafish retina, related to Figure 2. (A) Liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) chromatogram of 9-HSA and 10-HSA. The reverse phase chromatographic separation efficiency was evaluated by analyzing simultaneously 9-HSA and its isomer 10- HSA. (B) Quantification of 9-HSA endogenous amounts in zebrafish embryos of different developmental stages: prior to 1, 1, 3 and 4 days post-fertilization (dpf). An increase in the content of 9-HSA during development was observed. Results are expressed in μg/mg of lipid extract. (C – H) 9-HSA exogenous administration does not trigger apoptosis in the retina at 1, 2 and 3 days post-fertilization. DMSO –treated control (C, C’, E and G) and 9-HSA –treated retinae (D, D’, F and H) show no difference in the number of apoptotic cells at 1, 2 and 3 days post-fertilization (dpf) as revealed by a cell death TUNEL assay. (I – M) 9-HSA exogenous administration leads to an increase in proliferation in the retina from 1 day postfertilization. (I – J) Frontal confocal optical section of an immunohistochemistry of anti-pH3 (red) labeling cells in M-phase of the cell cycle on 24 hours post-fertilization (dpf) DMSO and 9-HSA injected embryos and counterstained with the nuclear marker DAPI (blue). (K) Quantification of the mitotic index at 24 hpf shows no difference in the number of pH3- positive cells in 9-HSA –treated retinae with respect to DMSO –treated control retinae (n = 6 for each condition. 56.33 SEM ± 0.1441 pH3-positive cells in DMSO control retinae and 54.5 SEM ± 2.895 pH3-positive cells in 9-HSA injected retinae, p-value = 0.6849, depicted as ns = non significant). An unpaired Welch’s t-test was used. (L – M) BrdU incorporation assay at 2 days post-fertilization (dpf) in DMSO-treated control and 9-HSA –treated embryos detected by immunohistochemistry after 12 hours shows a strong increase in the number of cells that actively replicated their DNA (S-phase, red) during these 12 hours. Nuclear counterstaining was also performed on these retinal sections by DAPI labeling (blue). Asterisk in (L) indicates unspecific antibody trapping in the lens. Scale bars: (C – F) = 20 μm, (I – J) = 50 μm, (L – M) = 20 μm.