Generation of Stable Cell Lines for Protein Expression

HeLa and RPE1 FlpIn cells were respectively grown in DMEM and DMEM/F12 supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml⁻¹), Ultra-glutamine (Sigma; 2 mM), blasticidin (4 μg ml⁻¹) and hygromycin for HeLa (200 μg ml⁻¹) or puromycin for RPE1 (1.6 μg ml⁻¹). 293Ts were grown in DMEM supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml⁻¹) and Ultra-glutamine (Sigma; 2 mM). Plasmids were transfected using Fugene HD (Roche) for HeLa or Lypofectamin LTX (Invitrogen) for RPE1 according to the manufacturer's instructions. To generate stably integrated HeLa and RPE1 FlpIn cell lines, pCDNA5-constructs were co-transfected with pOG44 recombinase in a 1:9 for HeLa and 1:5 ratio for RPE1 (ref. 55 (link)). Constructs were expressed by addition of 1 μg ml⁻¹ doxycycline for 24 h siHEC1 (custom; Thermo Fisher Scientific; 5′-CCCUGGGUCGUGUCAGGAA-3′) and siGAPDH (Thermo Fisher Scientific; D-001830-01-50) were transfected using HiPerfect (Qiagen) according to manufacturer's instructions.
Cells expressing RFP-MAD2 were obtained through lentiviral transduction and subsequent selection with puromycin (1.6 μg ml⁻¹).