Npc1 sirna

PC-12 cells (CRL1721) were purchased from ATCC and cultured at 37 °C in a humidified atmosphere containing 5% CO₂ in RPMI-1640 medium supplemented with 10% horse serum (Sigma, St. Louis, MI) and 5% fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were seeded in Poly-L-lysine (P1524, Sigma) coating glass bottom microwell dishes (MatTek Corporation, Ashland, MA) for two days before differentiation with nerve growth factor (300 ng/ml, Sigma) for 10 days. Npc1 siRNA (Strauss et al., 2010 (link)) (100 pmol, Qiagen, Valencia, CA) and scrambled control siRNA were transfected together with a GFP plasmid (2 μg) immediately before differentiation using the X-tremeGENE-siRNA transfection kit according to the manufacturer instructions (Roche). MMP-12 inhibitor (MMP408, 500 nM, Abcam) (Li et al., 2009a (link); Li et al., 2009b (link)) was added 4 h after transfection. Nine days after transfection, cells were fixed with 2% PFA for 15 min; cells were then examined and imaged using a fluorescence microscope (Nikon Eclipse TE2000-S) and a DS camera from Nikon. Neuritic length was measured as previously described using NIH ImageJ software (Das et al., 2004 (link)). Data were expressed as means of four sets of individual experiments; for each experiment at least 25–26 cells per group were analyzed and averaged values were used to calculate group means.