Generation of stable cell lines

The HEK293 Tet-On cell line (Flp-In T-Rex 293) purchased from Invitrogen (R780-07) was transfected with 2 μg of pcDNA5/RT/TO-NPAT and 10 μg of pOG44 using lipofectamine 2000 (Invitrogen) according to manufacturer instructions. Selection of stable cell lines was initiated 2 days after transfection using 75 μg/ml hygromycin. Twelve days after transfection, single colonies were isolated and cultured in 24-well plates. Cells were then expanded in 12-well and 6-well plates and finally in 25 cm² flasks. Induced expression of NPAT in the stable cells was verified by Western blotting using antibody sc32359.

SHSY-5Y cells were a kind gift from Professor Mike Ashford, University of Dundee. MISSION shRNA lentiviral transduction particles were used to Knock down NPAT in SHSY5Y cells. TRC1.5-pLKO.1-puro vector containing a hairpin insert with gene-specific sequence was used for cell transduction according to manufacturer protocols, in addition to hexadimethrine bromide (8μg/ml), to enhance transduction efficiency. Sequences of inserts in shRNA constructs targeting the NPAT gene are in S2 Table. MISSION PLKO.1-Puro-CMV-TurboGFP transduction particles (SHC003V) were used to monitor transduction efficiency. Transduced SHSY5Y cells were selected with 1 μg/ml puromycin and single cells were then plated in individual wells of a 96-well plate. Cells were expanded and the knockdown of NPAT expression was verified by Western blotting analysis.