Extensive endothelial autophagy was induced by the radiation in the brain. (A) Example of images (each representative of 6 zebrafishes) depicting autophagosome (green) of endothelial cells and autolysosome (red) of endothelial cells in 0 and 10 Gy group at 4-day post radiation. The right images are magnifications of the boxed areas in left images. Scale bars, 100 µm. (B) Morphometric analyses of GFP and mCherry double positive puncta (yellow puncta) and mCherry single positive puncta in 0, 5 and 10 Gy group at 4-day post radiation. The right images are magnifications of the boxed areas in left images. Scale bars, 100 µm. (C) Real-time quantification of single-cell mRNA expression levels of autophagy-related genes in radiation induced zebrafish endothelial cells at 2-day, 4-day post radiation respectively (n = 3 endothelial cells per group). Gene expression levels were normalized to those of β-actin in the control group. (D) Endothelial cells (flk:eGFP zebrafish lines) in vitro 3-D culture were gated for apoptosis staining in 0 and 10 Gy group at 4-day post radiation (n = 6 zebrafishes per group). Statistical analysis in (C) was performed using t-test: **P < 0.05, ***P < 0.01. Data represent the mean ± s.e.m.
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