Tet on gene expression system

TamR/TetOn-AAA cell line and the relative control (TamR/TetOn-V) were developed as previously described [9 (link)], using the Tet-On Gene Expression System (Clontech, Palo Alto, CA, USA). Briefly, TamR cells were first transfected with the regulator plasmid pTet-On, carrying the geneticin (G418) resistance gene, for the selection of successfully transfected cells. G418-resistant TamR/TetOn cells were, thus, isolated and further transfected with the pTRE-F3aAAA plasmid, bearing a cDNA encoding the entire open reading frame of a constitutively active form of the human FoxO3a gene. The zeocin resistance gene in the pTRE-F3aAAA plasmid assured proper selection of double transfected cells. Control cell lines (TamR/TetOn-V) were established following the same protocol but stably transfecting the pTRE backbone (vector only) in place of the F3aAAA cDNA insert. Pools of TamR/TetOn-AAA and TamR/TetOn-V cells were collected and cultured in TamR medium containing G418 and zeocin selection antibiotics, plus Tam 1 μM. Medium was replaced every 48 h. The tetracycline derivative doxycycline (Dox) (Sigma-Aldrich, Merck, Milan, Italy) was employed to induce F3aAAA expression.

TamR/TetOn-AAA cells and the corresponding control cell (TamR/TetOn-V) were generated using the Tet-On Gene Expression System (Clontech, Ancona, Italy). A detailed description of the cloning process can be found in the Supplementary Information.