Crl 1573

Human embryonic kidney (HEK-293, herein referred to as HEK; ATCC^®-CRL-1573^TM) cells and human hepatocellular carcinoma HepG2 (ATCC^®- HB-8065^TM) cells were obtained from ATCC (Manassas, VA). As we previously reported, both HEK and HepG2 cells do not express ABCB4^{9 (link),50 (link)}. Cells were grown in an incubator at 37 °C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (Gibco-Thermo Fisher Scientific, Villebon-sur-Yvette, France) containing 4.5 g/L D-glucose and supplemented with 10% heat-inactivated fetal bovine serum (Sigma, Saint-Quentin Fallavier, France), 2 mM L-glutamine, 2 mM sodium pyruvate, 100 units/mL of penicillin and 100 µg/mL streptomycin (Gibco-Thermo Fisher Scientific).
For transient transfection of HEK cells, they were seeded at subconfluent levels in the adequate tissue culture wells at least six hours before transfection. Turbofect (Thermo Fisher scientific) was used at a ratio of reagent:DNA of 2:1 according to manufacturer’s instructions. For transient transfection of HepG2 cells, subconfluent cultures were seeded in the adequate culture wells 24 hours before transfection. Lipofectamine 3000 (Thermo Fisher Scientific) was used at a ratio of reagent:DNA of 1.5:1 according to manufacturer’s instructions. Cell treatments and processing for further analyses were performed at least 16 hours post-transfection.

Human Embryonic Kidney HEK-293 (ATCC^®-CRL-1573^TM) cells and Human hepatocellular carcinoma HepG2 (ATCC^®-HB-8065^TM) cells were obtained from ATCC (Manassas, VA, USA). They were grown at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM), as previously reported [6 (link)]. Transfections with plasmids encoding ABCB4-wt or the mutants were performed using Turbofect at a ratio of reagent:DNA of 2:1 for HEK-293 cells, and JetPrime at a ratio of reagent:DNA of 2:1 for HepG2 cells, according to the manufacturer’s instructions and as previously described [6 (link)]. Stable expression in HEK-293 cells was obtained by selection with 400 µg/mL of G-418 sulfate (GE Healthcare, Chicago, IL, USA) for three weeks. Cells were subsequently grown in the presence of 100 µg/mL of G-418. For the experiments with HEK-293 cells, plates were precoated with 100 µL poly-L-lysine for 1 h at RT.

Human lung adenocarcinoma A549 cell line was obtained from American Type Culture Collection (ATCC, CCL-185™). Human colorectal adenocarcinoma DLD-1 (ATCC, CCL-221™) cell line was provided by Dr L Grumolato (DC2N laboratory, Inserm U1239, Mont-Saint-Aignan, France) and human embryonic kidney HEK-293 (ATCC, CRL1573™) cell line was generously given by Dr Prézeau (IGF laboratory, Montpellier, France). All cell lines were routinely maintained according to the instructions from ATCC. More precisely, A549 and DLD-1 cells were cultured with RPMI 1640 media and HEK-293 cells were cultured with DMEM media, all supplemented with 1% sodium pyruvate (ThermoFisher Scientific, Montigny-Le-Bretonneux, France) and 10% foetal bovine serum (FBS, Lonza, Levallois-Perret, France). Transient transfections were performed using either Amaxa^® Cell Line Nucleofactor^® Kit V (Lonza, Levallois-Perret, France) or FuGene^® HD (Promega Corporation, Southampton, UK) according to the manufacturer’s protocol.