The understandable lack of clinical clarification as

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The understandable lack of clinical clarification as to the nature of cell death involved in SMA justifies the need for both in vitro and in vivo mod- els susceptible to convergent cell viability assays. The objective of this study was to investigate in vitro how regulation of smn protein affected neu- ronal cell death using smn down-regulation by RNA interference and smn overexpression by adenoviral SMN gene transfer. The neuroblastoma hybrid (NSC-34) cell line (Cashman et al. , 1992), which resembles developing motor neurons, were used. Apoptosis was induced using staurosporine, a potent protein kinase-C (PKC) inhibitor (Cagnoli et al. , 1996). To study the effects of smn depletion, small inter- fering RNA (siRNA) technology (Trulzsch et al. , 2004; Girard et al. , 2006) was used to knock down SMN gene expression to levels similar to that observed in transgenic SMA mice (Monani et al. , 2000b). Quantitative real time PCR and western blot were used to confirm the down-regulation of SMN gene. The activation of the caspase cascade is a characteristic process in cells undergoing apoptosis (caspase-dependent pathway), and caspase-3 activa- tion is a major effector of neuronal cell death (for review, see Yakovlev and Faden, 2001). To study caspase-dependent apoptosis in smn-depleted cells, we used the cleavage of the chromophore p -nitroa- niline (pNA) to assay caspase-3 activity in NSC-34 cells following SMN specific or control siRNA transfection. Caspase-3 activity, a quantitative cell survival assay [3-(4,5-dimethyl thiazol-2-yl)-2,5- diphenyl tetrazolium bromide] (MTT) and an apop- tosis marker [(terminal deoxynucleotidyl trans- ferase-mediated dUTP nick-end labeling (TUNEL staining)] were used to investigate cell viability and apoptosis. Furthermore, adenoviral gene transfer was used to test whether overexpression of smn could mitigate staurosporine-induced cell toxicity.
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ANTI-APOPTOTIC EFFECT OF SMN ON NEURAL CELLS 41 MATERIALS AND METHODS NSC-34 Cell Culture The mouse NSC-34 cell line was produced by fus- ing motor neuron-rich mouse embryonic day 12-14 spinal cord cells with neuroblastoma N18TG2 (Cashman et al. , 1992). The cells were grown in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum with 100 units/ml penicillin and 100 µ g/ml strepto- mycin at 37ºC. The day before transfection, 2-4 x 10 5 cells per well were seeded on 6-well plates. Transfection was carried out using "stealth" siRNA (Invitrogen) according to the manufacturer's instruc- tions. Transfecting non-silencing, fluorescein- labeled control siRNA was used to optimize the transfection conditions. The siRNA concentration for each well was 100 nM. For the staurosporine studies, staurosporine was added to the medium 24 h after seeding the cells for a final concentration of 500 nM. Cell viability assays were performed after an additional 24 h incubation period.
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