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Fig. S2

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ZDB-IMAGE-190924-9
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Figures for Albadri et al., 2019
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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.

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Reprinted from Developmental Cell, 50(1), Albadri, S., Naso, F., Thauvin, M., Gauron, C., Parolin, C., Duroure, K., Vougny, J., Fiori, J., Boga, C., Vriz, S., Calonghi, N., Del Bene, F., Redox Signaling via Lipid Peroxidation Regulates Retinal Progenitor Cell Differentiation, 73-89.e6, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell