Amaxa nucleofector kit t

FOXO4-Flag overexpression constructs were generated as follows: FOXO4 cDNA was obtained from Addgene (plasmid 17549) and subcloned into a pCMV tag4C vector to generate pCMV tag4C FOXO4-Flag. BJAB was transfected with pCMV tag4C FOXO4-Flag or empty vector using the Amaxa Nucleofector™ kit T and the corresponding Amaxa Nucleofector™ System (Amaxa, Gaithersburg, MD). Stable cell lines overexpressing FOXO4 were selected with changes of fresh medium containing G418 (500 μg/ml) for 4 weeks. FOXO4-targeting and control siRNA were purchased from Dharmacon (Thermo Scientific, Waltham, MA).

HT1080 cells were maintained in Dulbecco’s modified eagle’s medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine and 1 $\times$ PenStrep. Cells were maintained in 10 or 15 cm TC-treated plastic dishes at $37{}^{\circ }$ C and 5 % ${\text{CO}}_2$ . HT1080 cells were transfected using Amaxa nucleofector Kit T, program L-005. Cells were transfected with 5 $\mathrm{\mu }$ g DNA (pcDNA3-Clover) following manufacturers guidelines and replated on 6 cm TC-treated plastic dishes overnight at $37{}^{\circ }$ C, 5% ${\text{CO}}_2$ . 35 mm glass bottom MatTek dishes that were coated with laminin 10 $\mathrm{\mu }$ g/ml diluted in PBS and left overnight at $4{}^{\circ }$ C. Cells were then collected and replated onto the dishes and incubated for 24 hours at $37{}^{\circ }$ C, 5 % ${\text{CO}}_2$ . After, the dishes were washed twice with PBS before fixing with 4 % Paraformaldehyde for 10 min. These were then washed three times with PBS before slight drying. 10 $\mathrm{\mu }$ l Fluromount-G (without DAPI) was added on top of the cells and a 19 mm glass coverslip was added on top to seal the cells in the dish. Dishes were kept in the dark until imaging.