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and drug resistant cancer cells express an increased level of AC which
has been known to be functionally important for cancer cell prolifera-
tion and has hence been proposed as an attractive target for cancer
therapy.21,22 Our results from single cell gene expression analysis and
neurosphere assay with AC inhibitors imply that AC is one of the
important molecules highly expressed to render the NSPCs more
proliferative in the early stage of neurosphere formation.
In conclusion, we developed a novel fluorescent chemical
probe CDy5 that stains NSPCs in heterogeneous population of
cells in a neurosphere by binding to AC which was revealed in
this study as an important molecule for NSPCs to proliferate and
form neurospheres. Our work described here will provide an
invaluable tool to understand mammalian neural development.
We thank Dr Clement Khaw (SBIC-Nikon Imaging Centre)
for help with confocal microscopy. This study was supported by
an intramural funding from A*STAR (Agency for Science,
Technology and Research, Singapore) Biomedical Research
Council and a Singapore Ministry of Education Academic
Research Fund Tier 2 (MOE2010-T2-1-025).
Notes and references
Fig. 3 (a) Fluorescence image of CDy5-stained neurosphere protein extract
separated by isoelectric focusing (pH 3–10) and SDS-PAGE. The major fluorescent
spots of B35 kDa were marked with a rectangle (left upper panel) and magnified
(left lower panel). The proteins in the gel were detected by silver staining (right
panel). (b) western blot of CDy5-labeled protein with the acid ceramidase (AC)
antibody. The CDy5 signal (upper panel, 532/580 nm) and a signal from the
secondary antibody that recognizes the AC antibody (middle panel, 633/670 nm)
overlapped (lower panel). (c) Pull-down assay of CDy5-labeled protein using the
acid ceramidase antibody (AC Ab). (d) Fluorescence image of CDy5-stained
neurosphere protein extracts separated by SDS-PAGE. Control 1, protein extract
in lysis buffer; control 2, protein extract in enzyme reaction buffer.
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7494 | Chem. Commun., 2014, 50, 7492--7494
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