All constructs were tested in HEK293T human embryonic kidney, P19 mouse embryonal carcinoma, and SH-SY5Y human neuroblastoma cells. Cell culture conditions, including growth and maintenance have been carried out as described in Mexal et al. (Mexal et al., 2007). Transfections were carried out by seeding 0.5×106 cells per well in a 24 well plate. The following day cells were transfected using FuGENE® HD tranfection reagent (Roche) with 0.2 ug of the test plasmids. Approximately 5–10ng of a control vector (pRL-CMV; renilla luciferase) was co-transfected to control for transfection efficiency and prevent competition of the test and control plasmids for transcription factor binding SH-SY5Y cells were induced to differentiate by incubation with 75mM all-trans-retinoic acid in addition to normal growth media for 10 days prior to transfection.
