Stable Cell Lines for Cellular and Molecular Biology Research

HeLa and HEK293 Flp-In Trex (Life Technologies) cells were grown in DMEM supplemented with Glutamax, 9% heat-inactivated fetal calf serum and 1 × penicillin and streptomycin (Life Technologies). The stable HeLa Kyoto cell line expressing GFP-tubulin and H2B-mCherry20 (link) was maintained in the presence of 2 μg ml⁻¹ puromycin (Carl Roth GmbH) and 500 μg ml⁻¹ G418 (Sigma) until transfection. The stable HeLa cell line expressing GFP-centrin41 (link) was maintained in the presence of 500 μg ml⁻¹ G418 (Sigma).
A stable HEK293 cell line expressing FLAG-MCRS1 was generated by cotransfection of the pFlag-MCRS1 construct25 (link) and the pBABE-puro vector (Addgene). Clones were selected using puromycin (2.5 μg ml⁻¹; Sigma-Aldrich) and selected for FLAG-MCRS1 expression by western blot.
Stable inducible HEK293 Flp-In Trex (Life Technologies) cell lines carrying KANSL1-FBH (3XFLAG/biotin acceptor site/6XHis), KANSL3-FBH and MCRS1-FBH were generated by flippase recognition target (FRT) recombination according to the product manual. These cell lines were maintained in the presence of 15 μg ml⁻¹ blasticidin and 150 μg ml⁻¹ hygromycin. The day before synchronization, cell lines were induced by addition of 100 ng ml⁻¹ doxycycline.