DNA Transfections

HER.TLR^TetO.KRAB cells were kept for 10 days in medium lacking or containing Dox at a final concentration of 0.5 μg mL^–1. Next, each of these cell cultures (i.e., with and without Dox) were seeded 1 day before DNA transfections in wells of 24-well plates (Greiner Bio-One) (Tables S2–S7; Figure S4A). The DNA transfections were initiated by adding 1 mg mL^–1 linear 25 kDa polyethyleneimine (PEI; Polysciences) to the different plasmid mixtures diluted in 50 μL 150 mM NaCl (Tables S2–S7; Figure S4A). After vortexing for 10 s, the DNA-PEI complexes were let to be formed for 15 min at room temperature, after which they were directly added to the medium of the cell cultures. The different transfection mixtures were substituted 6–8 h later by regular culture medium with or without Dox. At 3 days post-transfection, the cells were sub-cultured every 3–4 days for a period of 11 days, and the frequencies of EGFP- and mCherry-positive cells in the cultures were determined by flow cytometry (Figure S4A). To activate transgene expression, the cultures initially lacking Dox were exposed to Dox (0.5 μg mL^–1) for 10 days, after which the frequencies of EGFP- and mCherry-positive cells were also determined in these cultures by flow cytometry (Figure S4A). The transfection protocols and experimental design applied to the control TetO-negative HER.TLR^KRAB cells were similar to those applied to the TetO-positive HER.TLR^TetO.KRAB cells (Table S5; Figure S4B).

HEK.EGFP^TetO.KRAB cells were cultured for 7 days in the presence or absence of Dox at a final concentration of 0.2 μg mL^–1. Next, the cells were seeded 1 day before DNA transfections in wells of 24-well plates (Greiner Bio-One) (Tables S8–S10; Figure S4C). The DNA transfections started by adding 1 mg mL^–1 PEI to the different plasmid mixtures diluted in 50 μL 150 mM NaCl (Tables S8–S10; Figure S4C). After vortexing for 10 s, the DNA-PEI complexes were let to be formed for 15 min at room temperature, after which they were directly added to the medium of the cell cultures. The various transfection mixtures were replaced 6–8 h later by regular culture medium with or without Dox. At 3 days post-transfection, the cells were sub-cultured every circa 3 days for a period of 7 days, and the frequencies of EBFP-positive and EGFP-negative cells in the cultures containing Dox were determined by flow cytometry (Figure S4C). To activate transgene expression, the cultures that initially had not received Dox were incubated in the presence of Dox (0.2 μg mL^–1) for an additional 7-day period, after which the frequencies of EBFP-positive and EGFP-negative cells were also determined in these cultures by flow cytometry (Figure S4C).