Pcdna3

Human MAO-A was stably over expressed in the human neuroblastoma SH-SY5Y cell line by cloning human MAO-A cDNA into the expression vector (pcDNA3.1-, Invitrogen, Karlsruhe, Germany), which was then transformed into competent bacteria. The expressed plasmid DNA was purified using DNA mini and DNA midi kits (Qiagen, Manchester, UK) and SH-SY5Y cells were transfected with 1 µg of pcDNA3.1(-) containing the hMAO-A insert or 1 µg of the empty pcDNA3.1(-) vector via electroporation using the Amaxa nucleofection system (Amaxa, Cologne, Germany). Electroporated SH-SY5Y cells were seeded in 6-well tissue culture plates in normal growth medium, which was replaced after 24 h. 48 h post-transfection, cells were passaged and seeded at 25% confluence in growth medium containing G418 sulphate (geneticin) at a concentration of 700 µg/ml. Stable SH-SY5Y clones were selected by their resistance to geneticin, which is conferred by the plasmid vector pcDNA3.1. The medium was replaced every 3–4 days until resistant foci were visible. Resistant cells were diluted in 96 well plates at a density of 0.5 cell/well and single clones were expanded and assayed for MAO-A expression.

PLAC2 and PTEN expression vectors were constructed using pcDNA3 (Sangon Biotech Co., Ltd.). PTEN small interfering (si)RNA (5′-UAGCAGAAACAAAAGGAGAUAUC-3′) and negative control siRNA (5′-GUCGUCAAAGUCAGGUACACCGA-3′) were from Shanghai GenePharma Co., Ltd. Y79 and WERI-Rb-1 cells were collected at the confluence of 70–80%. Nucleofector™ Technology (Lonza Group, Ltd.) was used to transfect 10 nM PLAC2 and PTEN expression vector, 10 empty pcDNA3 vectors negative control (NC), 35 nM PTEN siRNA, or 35 nM NC siRNA were transfected into 10⁵ cells. The control group included cells with no transfections. Subsequent experiments were performed at 24 h post-transfections.