Expression was calculated by comparative ct method

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Expression was calculated by comparative Ct method with cDNA sample quantification normalized to β -actin expres- sion. B: Semi-quantitative analysis of smn protein by western blot assay. The density of bands was compared to β -actin. Densitometric analysis of Western blot revealed that protein levels of smn decreased from 47.5% of control at 48 h to 42% at 72 h. FIGURE 2 NSC-34 cell viability after SMN siRNA transfection. A: Relative viability assessed by MTT reduction in NSC-34 cells ( N =8: *** p <0.005; **** p <0.0001). B: Caspase-3 activation in NSC-34 cells. Background corrected colorimetric measurements of chromophore cleavage were transformed against a pNA titration curve (y = 0.0025x - 0.0013) and presented as percentages ( N =4: n.s. p =0.148; ** p <0.01).
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G.C. PARKER et al. 44 scrambled siRNA ( p <0.01). Densitometric analysis of Western blot revealed that protein levels of smn decreased from 47.5% of control at 48 h, to 42% of control at 72 h (FIG. 1B). NSC Viability Following SMN siRNA Transfection One index of cell viability is mitochondrial enzyme activity. The ability of neural cells to reduce MTT by mitochondrial dehydrogenases was significantly decreased relative to control by 13% at 48 h after siRNA transfection ( p <0.005, FIG. 2A). By 72 h, the viability of smn-depleted cells was decreased by 23% compared to scrambled control ( p <0.0001). In contrast, control siRNA transfected cells showed no significant decrease in cell viability at 72 h ( p =0.719). The results from this assay suggest that the cell viability of NSC-34 cells is negatively affected by the loss of smn. Caspase-3 activity was assayed using cleavage of the chromophore p -nitroaniline (pNA) in NSC-34 cells following SMN specific or control siRNA transfection. Background corrected readings were transformed against a pNA standard titration curve (y = 0.0025x - 0.0013), then presented as percent- ages (FIG. 2B). Caspase-3 activity had decreased by 72 h in control cells. The increase in caspase-3 activity at 48 h only approached significance ( p =0.148), while caspase-3 activity remained ele- vated in SMN siRNA transfected NSC at 72 h rela- tive to control ( p <0.01). A standard TUNEL staining for DNA fragmenta- FIGURE 3 DNA fragmentation in SMN SiRNA trans- fected NSC cells. The incidence of apoptosis in NSC-34 cells was assessed by counting TUNEL-positive and -negative cells at 48 and 72 h after transfection with control or SMN siRNA. Transfection with SMN siRNA increased significantly the percentage of TUNEL- positive cells at 48 and 72 h ( N =5, 6: * p <0.05; *** p <0.001, compared to respective controls). Upper panels show representative cell staining. FIGURE 4 NSC-34 cell viability exposed to staurosporine and smn overexpression. A: Relative viability assessed by MTT reduction in NSC-34 cells, exposed to an apoptosis-inducing concentration of staurosporine (500 nM), previ- ously treated with either a control (LacZ) or SMN adenoviral vector (N=9: **** p <0.0001 compared to control or Stau + AdV.SMN). B: Caspase-3 activation in similarly treated NSC-34 cells ( N =9: **** p <0.0001 compared to DNase or Stau). Measurement details as in Figure 2B.
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ANTI-APOPTOTIC EFFECT OF SMN ON NEURAL CELLS 45 tion was performed to study the rate of apoptotic cell death. Three per cent of NSC-34 cells trans-
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