*4.5. Statistical Analyses*

Graphics and one-way ANOVA tests were performed using Prism version 7.00 (Graph-Pad Software, La Jolla, CA, USA). A *p* value of less than 0.05 was considered significant with \*: *p* < 0.05; \*\*: *p* < 0.01; \*\*\*: *p* < 0.005; ns: not significant. Symbols always indicate the comparison between the control and the other tested conditions.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22137087/s1. Figure S1: Co-immunoprecipitation of ABCB4 and RAB10 in HEK cells. HEK cells co-expressing RAB10-GFP and ABCB4 were used. GFP (A) and ABCB4 (B) were immunoprecipitated using specific antibodies. Controls were performed using unspecific antibodies (unspe.). After SDS-PAGE, the presence of ABCB4 and RAB10-GFP in the lysates (Input) and the immunoprecipitates (IP) was detected by immunoblot (IB) as indicated. Molecular weight markers (in kDa) are shown. These panels are representative of three independent experiments per condition. Figure S2: Full immunoblots related to Figure 1A,B. Results shown in Figure 1A,B are delineated by dotted rectangles. MW (in kDa) are indicated. Figure S3: Phosphatidylcholine efflux after RAB10-WT expression. The phosphatidylcholine efflux from HEK cells expressing ABCB4-WT or RAB10-WT-GFP was measured as in Figure 2C. Note that for these experiments, means could not be normalized to ABCB4 expression levels. Means (± SD) of three independent experiments are shown. Figure S4: Full immunoblots related to Figure 2A. Results shown in Figure 2A are delineated by dotted rectangles. MW (in kDa) are indicated. Figure S5: ABCB4 immunolocalization in HepG2 cells. ABCB4 and the indicated forms of RAB10-GFP (GFP alone as control, ctrl) were co-expressed in

HepG2 cells. Total ABCB4 and RAB10-GFP localization was analyzed as in Figure 3B. This figure is representative of three independent experiments. Bars: 10 µm. Figure S6: ABCB4-I541F-FLAG immunolocalization in HeLa cells. ABCB4-WT-FLAG or ABCB4-I541F-FLAG were expressed in HeLa cells. After treatment with vehicle (DMSO) or 10 µM cyclosporin A, cell surface and total ABCB4 were immunolabeled and visualized as in Figure 3B. This figure is representative of three independent experiments. Bars: 10 µm. Figure S7: Full immunoblots related to Figures 4A and 5A. Results shown in Figures 4A and 5A are delineated by dotted rectangles. MW (in kDa) are indicated. Figure S8: ABCB4 expression after RAB10 knockdown. Cell lysates from HeLa cells (A) and HEK cells (B) treated as in Figures 4 and 5, respectively, were analyzed by immunoblot using the indicated antibodies (upper panels). MW (in kDa) are indicated. These panels are representative of three independent experiments per condition. ABCB4 signal intensities were quantified and means (± SD) of three independent experiments are shown (lower panels). Figure S9: Confocal microscopy analysis of RAB10-WT subcellular localization in HeLa cells. HeLa cells were transfected with a RAB10-WT-GFP-encoding construct (green) and the following intracellular compartments were immunolabeled (red): calnexin for endoplasmic reticulum, giantin for the Golgi apparatus, EAA1 for early endosomes, LAMP1 for late endosomes and lysosomes. This figure is representative of three independent experiments. Bars: 10 µm.

**Author Contributions:** A.B.S., V.V., and T.F. designed the study. A.B.S., V.V., E.B., A.-M.D.-S. and T.F. performed the experiments. A.B.S., V.V., M.L., E.M., A.-M.D.-S., A.B., J.-L.D., E.G., C.H., T.A.-S., F.G., E.J. and T.F. analyzed the data and provided significant intellectual contribution. A.B.S. and T.F. wrote the manuscript, which was reviewed and approved by all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** A.B.S. and E.M. were supported by the « Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation ». T.F. was supported by grants from the Agence Nationale de la Recherche (ANR-15-CE14-0008-01), the French Association for the Study of the Liver (AFEF) and FILFOIE (Filière de santé des maladies rares du foie, Paris, France). T.A.-S. was supported by grants from the Association Mucoviscidose-ABCF2, Fondation pour la Recherche Médicale (EQU2020- 03010517) and FILFOIE. The Orbitrap Fusion mass spectrometer was acquired with funds from the FEDER through the «Operational Programme for Competitiveness Factors and employment 2007-2013», and from the «Cancéropôle Ile-de-France».

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** We are grateful to FILFOIE, AMFE (Association Maladie Foie Enfants, Malakoff, France), MLD (Monaco Liver Disorder, Monaco), Association "Pour Louis 1000 Foie Merci" (Fournet-Luisans, France), Association "Il était un foie" (Plouescat, France) and Fondation Rumsey-Cartier (Genève, Switzerland) for their financial support. We also thank Mark McNiven (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, USA) for providing RAB10-encoding plasmids, Wadih Ghattas and Charles Skarbek (Institut de Chimie Moléculaire et des Matériaux d'Orsay, Université Paris-Saclay, Orsay, France) for their help with multiplate fluorescence reading, as well as Lynda Aoudjehane (Human HepCell–ICAN, Saint-Antoine Research Center, Paris, France) for providing primary human hepatocytes, Jérémie Gautheron (Saint-Antoine Research Center, Paris, France) for his help with the CRISPR/Cas9 strategy and Isabelle Garcin (UMR\_S 1193 Inserm/Université Paris-Saclay, Orsay, France) for her help with imaging studies.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
