*2.3. RAB10 Silencing Reduces ABCB4 Plasma Membrane Expression and Decreases Its Function*

Then, we investigated the effect of RAB10 silencing on ABCB4 cell surface expression using a siRNA approach. The transfection of specific anti-RAB10 siRNAs in HeLa cells induced an important knockdown of endogenous RAB10 (Figure 4A). The quantification of these experiments indicated that RAB10 expression was decreased by more than 90% in comparison with cells transfected with control siRNAs (Figure 4B). In this RAB10 knock-

down condition, using the specific plasma membrane labeling described above, we observed that less ABCB4 was localized at the plasma membrane than in the control condition (Figure 4C), which was confirmed by the quantification of these experiments (Figure 4D).

**Figure 4.** Effect of RAB10 knockdown on ABCB4 localization in HeLa cells. (**A**) Forty-eight hours after siRNA transfection in HeLa cells (Control, ctrl or anti-RAB10), RAB10 expression was analyzed by immunoblot, as in Figure 2A. This panel is representative of three independent experiments. The presented data were cropped from full immunoblots shown in Supplementary Figure S7. (**B**) Densitometry analysis of (A), as performed in Figure 2B. Means (±SD) of at least three independent experiments are shown; \*\*\* *p* < 0.005. (**C**) HeLa cells treated as in (A) were used to analyze ABCB4- FLAG localization at the plasma membrane, as performed in Figure 3B. This panel is representative of three independent experiments. Bars: 10 µm. (**D**) Quantification of (C), as performed in Figure 3C. Means (±SD) are represented; \*\*\* *p* < 0.005.

To confirm the effect of RAB10 silencing on ABCB4 cell surface expression, we used RAB10-KO HEK cells generated by the CRISPR/Cas9 approach. This strategy was complementary to siRNA transfection and less aggressive for the cells in the frame of assays aiming at measuring ABCB4-mediated PC secretion. Immunoblot analyses of RAB10 expression indicated an important knockdown of protein expression in HEK cells (Figure 5A) to more than 90% compared to control cells (Figure 5B). Then, ABCB4 cell surface expression was determined as aforementioned. In line with our previous results in HeLa cells, an important decrease in ABCB4 cell surface expression was detected in RAB10-KO HEK cells, in comparison to control cells (Figure 5C; quantification in Figure 5D). We also examined the effect of RAB10 silencing by CRISPR/Cas9 on ABCB4 function in HEK cells: ABCB4 mediated PC secretion was strongly impaired in RAB10-KO cells in comparison to control cells (Figure 5E). Altogether, these results indicate that RAB10 knockdown diminishes plasma membrane expression levels of ABCB4, and thus indirectly its PC secretion function towards the extracellular environment.

**Figure 5.** Effect of RAB10 knockdown on ABCB4 localization and function in HEK cells. (**A**,**B**) RAB10 expression in control (ctrl) and RAB10-KO HEK cells was analyzed (A) and quantified (B) as in Figure 4A,B. (A) is representative of three independent experiments and (B) represents the means (±SD) of three independent experiments; \*\*\* *p* < 0.005. The presented data were cropped from full immunoblots shown in Supplementary Figure S7. (**C**) HEK cells treated as in (A) were used to analyze ABCB4-FLAG localization at the plasma membrane, as performed in Figure 3B. This panel is representative of three independent experiments. Bars: 10 µm. (**D**) Quantification of (C), as performed in Figure 3C. Means (±SD) are represented; \*\*\* *p* < 0.005. (**E**) Control (ctrl) and RAB10-KO HEK cells were used to measure ABCB4-mediated PC secretion, as performed in Figure 2C. Means (±SD) of three independent experiments are shown; \* *p* < 0.05.
