*2.4. RAB10 Promotes ABCB4 Trafficking from the Golgi Apparatus to the Plasma Membrane*

Since we observed a considerable reduction of ABCB4 expression at the cell surface after RAB10 knockdown, both in HeLa and HEK cells (see Figures 4 and 5) without significant modification of total ABCB4 expression (Supplementary Figure S8), we inferred that RAB10 knockdown caused an intracellular retention of ABCB4. As previously reported [22], we found that RAB10 was preferentially associated with the Golgi apparatus, colocalizing with giantin (Supplementary Figure S9). We thus hypothesized that the plasma membrane targeting of ABCB4 might be impaired at post-Golgi steps when RAB10 is knocked down. The total ABCB4 distribution was examined by confocal microscopy in both HeLa and HEK cells. Under control conditions, ABCB4 displayed a significant plasma membrane staining (Figure 6A,B, upper panels). However, after RAB10 knockdown, ABCB4 was less present at the plasma membrane with an increased colocalization with the Golgi marker giantin, both in HeLa cells (Figure 6A, lower panels) and HEK cells (Figure 6B, lower panels). These results provide evidence that RAB10 is involved in ABCB4 trafficking from the Golgi apparatus to the plasma membrane.

**Figure 6.** Effect of RAB10 depletion on total ABCB4 subcellular distribution. (**A**,**B**) HeLa cells transfected with control (si-ctrl) or anti-RAB10 (si-RAB10) (A), and control (ctrl) vs. RAB10-KO HEK cells (B) were used to analyze ABCB4 localization by indirect immunofluorescence and confocal microscopy. ABCB4 (red) and giantin (green) were labeled using specific antibodies while nuclei were stained with Hoechst 33,342 (blue). Each panel is representative of at least three independent experiments per condition. White arrows indicate colocalization of ABCB4 with giantin at the Golgi apparatus. Bars: 10 µm.
