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Fig. 4. IRS1 is necessary to maintain CGNP proliferation. (A) CGNPs
were treated with SHH for 24 hours prior to infection with IRS1 shRNAs for 3
hours at which time the medium was replaced with either SHH treated or
untreated medium. Treatment with shRNAs knocked down levels of IRS1 (red box)
as well as levels of cyclin D2 without affecting the activation of AKT or
levels of IRS2. β-Tubulin confirms equal loading. (B) Infection of
cerebellar slices with lentiviruses targeting IRS1 causes reduced thickness of
the EGL (broken white lines) and reduced BrdU incorporation (red) in the EGL.
Blue represents DAPI counterstaining. (C) Levels of proliferation in
primary CGNP cultures in response to IRS1 shRNA lentivirus infection were
determined by measuring BrdU incorporation after a 2-hour BrdU pulse. Reduced
BrdU incorporation is evident in shRNA plus SHH compared with SHH-treated
CGNPs. BrdU-positive cells were counted and values expressed as percent of
total cells per field. Values are represented as fold BrdU-positive cells over
untreated CGNPs. *P<0.05, n=4. (D)
Levels of BrdU incorporation were assessed as above in Percoll
gradient-purified CGNP cultures. Trends of BrdU incorporation remain the same
as mixed cultures. **P<0.01. (E) Cell survival
was assessed by immunostaining for cleaved caspase 3. Based on quantification
of cleaved caspase 3-positive cells, there was no change in cell survival
regardless of treatment. (F) Cell survival in response to shRNA
treatment remained the same in purified cultures. Cleaved caspase 3-positive
cells were counted and values are expressed as percent of total cells per
field. Error bars represent s.e.m., n=5.
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