Pegfp n1 construct

Cells were cultured in a 6-well plate and allowed to adhere overnight before transfection with either a negative control siRNA, or MYC siRNA at a concentration of 50 nM using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The sequences for the MYC siRNA and control siRNA were previously described [21 (link)]. The transfection efficiency was judged by expression of a co-transfected pEGFP-N1 construct (Clontech, now Takara Bio, Mountainview, CA).

The human LG72 cDNA (GenBank sequence: AY138546) cloned into the pEGFPN1 vector was a generous gift from the Institute of Molecular Psychiatry, University of Bonn, Germany (Otte et al., 2011 (link)), and the pEGFPN1 construct (Takara Clontech) was used as a control plasmid. The human DAO cDNA (GenBank sequence: BC029057) cloned into pCMV3-c-Myc vector (HG13372-CM, Sino Biological) and the pCMV3-c-Myc vector (CV014, Sino Biological) were used for transfection. For all experiments except cell viability, HEK293, 1321N1 and SH-SY5Y cells were seeded into 6-well plates (3335, Corning) at a density of 2 × 10⁵ cells, 5 × 10⁵ and 1 × 10⁶ cells in 1 mL growth medium, respectively. The cells were allowed to adhere for 24 h. The cells were transfected with 5 μg of above-mentioned plasmids using Xfect transfection reagent (631317, Takara Clontech) according to manufacturer’s guidelines. The growth medium was exchanged 4 h after transfection. For all experiments, cells were incubated for 48 h following transfection.