Decreased Ngb expression exacerbates hypoxic neuronal death.
(a) Representative Western blot showing decreased Ngb
expression compared to untransfected control cells (Con) in cultured
cortical neurons treated with an antisense ODN directed against Ngb,
but not with a sense ODN (Left). Ngb expression was
quantified (mean ± SEM, n = 3) by computer
densitometry (*, P < 0.05 relative to Con
by t test) (Right). (b)
Cell viability, measured by MTT absorbance or TBE, in cultures
maintained for 12 h without oxygen or in the presence of 0.1 μM
staurosporine (Stauro) or 200–400 μM SNP, under standard conditions
(no ODN) or after treatment with 5 μM sense or antisense ODN, added
3 h before the onset of, and present throughout the toxic exposure
(n = 3–6). *, P < 0.05
relative to no treatment (t test). (c)
Fluorescence labeling of cultured cortical neurons treated with Ngb
antisense (Upper) or sense (Lower) ODNs,
showing immunoreactivity for the 17–20-kDa caspase-3 cleavage product
(Left, red), ODN fluorescence (Center,
green), and the merged images (Right, yellow). (Original
magnification, ×400). Antisense-transfected cultures show an increase
in the proportion of neurons that exhibit caspase-3 cleavage compared
to sense-transfected cultures, consistent with the antisense-mediated
decrease in cell viability shown in b.