*4.1. Plasmids, Cell Culture and Transfection*

Two different constructs encoding human ABCB4 were used: pcDNA3-ABCB4, which was previously described [28], and pcDNA3-ABCB4-FLAG, a modified version of ABCB4 with a FLAG tag (DYKDDDDK) within its first extracellular loop (between Ser 99 and Leu 100), prepared by Genscript (Piscataway, NJ, USA). Constructs encoding human RAB10- WT (pEGFP-RAB10-WT) and its constitutively active (pEGFP-RAB10-Q68L) and inactive (pEGFP-RAB10-T23N) forms, all with N-terminal GFP, were a kind gift from Mark McNiven (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA), which were prepared as described [43].

Primary human hepatocytes were prepared as described [44] and kindly provided by the Human HepCell platform (ICAN, Paris, France). Human embryonic kidney cells (HEK-293, herein referred to as HEK; ATCC®-CRL-1573TM) and HeLa cells (ATTC®-CCL-2TM) were grown at 37 ◦C with 5% CO<sup>2</sup> as described [45]. HEK and HeLa cells were transfected using Turbofect (Thermo Fisher Scientific, Villebon-sur-Yvette, France) at a ratio of reagent:DNA of 2:1 according to the manufacturer's instructions. JetPrime (PolyPlus Transfection, Illkirch, France) was used for small interfering RNA (siRNA) transfection according to the manufacturer's instructions.
