CAD Mouse Neuroblastoma Cell Culture

CAD mouse neuroblastoma cells (42 (link)) were cultured in Dulbecco's Modified Eagle Medium/High Glucose (DMEM; Thermo Fisher Scientific/GIBCO), supplemented with 11% FetalClone III Serum (GE Healthcare/HyClone), 1 mM sodium pyruvate (Thermo Fisher Scientific/GIBCO), 100 IU/ml penicillin and 100 μg/ml streptomycin, at 37°C in the presence of 5% CO₂. For transfection experiments, cells were plated in the CAD medium without antibiotics at a density of 4 × 10⁵ cells per well of a six-well plate for tissue culture. Twelve hours post-plating, cells were transfected with corresponding ON-TARGETplus siRNA (GE Healthcare/Dharmacon; siControl [D-001810-01-20]; siPtbp1 [J-042865-11-0050]; siPtbp2 [L-049626-01-0005]) using Lipofectamine RNAiMAX (Thermo Fisher Scientific/Invitrogen). Following 36-hour incubation, cell cultures were typically re-transfected with 1 μg of a minigene plasmid using Lipofectamine 2000 and incubated for another 36 h prior to RNA harvest. For U1 snRNA suppressor experiments, the ratio of pN/S6U1 plasmid to minigene plasmid used for transfections is 9:1. Neurons, neuronal progenitor cells and astrocytes were isolated from mouse cortices and maintained as described elsewhere (43 (link)).