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Supplementary Figure 1 Apoptosis of neural progenitors after irradiation is dependent on p53
status. Apoptotic cells in the subgranular zone (SGZ) of the dentate gyrus (a−f) are rarely
observed in non-irradiated mice (a, c, e). At 8 hours after 17 Gy, numerous apoptotic cells with
characteristic nuclear condensation and fragmentation using DAPI nuclear staining are seen in
the SGZ (b) of p53 wildtype (+/+) mice. Mice heterozygous () for p53 appear to have an
intermediate response (d) after 17 Gy, and the response was not observed in mice knockout ()
of the p53 gene (f). The number of apoptotic cells at 8 hours after 17 is radiation dose and p53
status dependent (table).
Supplementary Figure 2 Radiation-induced apoptosis of endothelial cells in mouse brain is
regulated by smpd1 gene. A TUNEL positive cell (arrow, a) is associated with a CD31-labeled
microvessel endothelial cell (arrow, b; merged, c) in the mouse dentate gyrus at 8 h after 17 Gy.
The number of CD31 positive apoptotic cells in mouse dentate gyrus at 8 h after irradiation is
radiation dose (P<0.001, two-way ANOVA) and smpd1 genotype (P<0.001) dependent (d).
Error bars represent means ± S.E.M.
Supplementary Figure 3 Cells dissociated from neurospheres cultured from mouse brain
demonstrate phenotypic markers of neural progenitors. They show dual immunostaining for
nestin (a) and Sox2 (b; DAPI, c; merged, d). These cells also show immunoreactivity for
musashi-1 (e; DAPI, f; merged, g), phenotypic markers of neural progenitors.
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Supplementary Figure 4 Neural progenitors cultured from smpd1+/+, smpd1−/−, p53+/+ and
p53−/− mice demonstrate multipotential ability and are able to differentiate in vitro into
oligodendrocytes, astrocytes and neurons as revealed by immunocytochemistry (red) for
galactocerebroside (GC, a−d), GFAP (e−h) and MAP2 (i−l) respectively. DAPI nuclear
counterstaining is shown in blue.
Supplementary Figure 5 Neural progenitors undergo apoptosis in vitro after irradiation. At 24
hours after 5 Gy, musashi (a) and Sox 2 (d) stained cells demonstrate characteristic nuclear
condensation and fragmentation of apoptosis as shown by DAPI nuclear countstaining (arrows, b
and e; merged, c and f)
Supplementary Figure 6 Neural progenitors cultured from eGFP mice demonstrate
multipotential ability and can differentiate into oligodendrocytes (green, eGFP, a; red, GC, b),
astrocytes (eGFP, c; GFAP, d) and neurons (eGFP, e, g; MAP2, f; -III tubulin, tub III, h).
Nuclei are counterstained using DAPI.
Supplementary Figure 7 Neural progenitors from eGFP mice undergo apoptosis in vitro after
irradiation. Compared to non-irradiated cells (a−c), a single dose of 5 Gy induces many
apoptotic cells (d−f) that show characteristic nuclear condensation upon DAPI (e), and are also
positive for TUNEL (f). A single dose of 5 Gy induces a significant increase in the number of
apoptotic cells in neural progenitors cultured from eGFP mice (g). Results represented average of
a minimum of 3 experiments, and a minimum of 500 cells counted per experiment. Error bars
represent means ± S.E.M., * P<0.01, t-test, compared to non-irradiated controls.
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Supplementary Figure 8 Neural progenitors from eGFP mice transplanted stereotactically into
the mouse hippocampus are able to differentiate into neurons. A transplanted eGFP cell (arrow,
a) demonstrates the neural progenitor phenotypic marker nestin (b; DAPI, c; merged, d). Two
eGFP cells in the dentate gyrus (arrows, e) show immunostaining for DCX (arrows, f), a marker
for neuronal progenitors (DAPI, g; merged, h), and an eGFP cell (arrow, i) has differentiated
into a neuron and demonstrates immunoreactivity for the neuronal marker NeuN (arrow, j;
DAPI, k; merged, l).
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