Rab18 contributes to directional bias of cell migration during chemotaxis. (a) Chemotaxis of MDA-MB-231 cells treated with control siRNA, siRNA against Rab18, or silenced for Rab18 and then transfected with GFP-Rab18, toward FCS. Cells were imaged in a chemotaxis chamber at 10-min intervals over a 2-h period. Direction of the chemoattractant gradient is toward the bottom. Rose plots and Rayleigh test show that the migration of control but not Rab18 knockdown cells is directionally biased. A total of 163 control cells, 194 Rab18 knockdown cells, and 73 cells silenced for Rab18 and then transfected with GFP-Rab18 from three independent experiments were quantified. (b) Quantification of the forward migration index in the x- and y-direction for the same cells as in panel a. *, P < 0.05; **, P < 0.01. (c) Transmission images of MDA-MB-231 cells treated with control siRNA, siRNA against Rab18, or silenced for Rab18 and then transfected with GFP-Rab18 (GFP channel in the inset) during chemotaxis. The direction of the chemoattractant gradient is toward the bottom. Scale bar: 10 µm. (d) Percentage of cells having one protrusion oriented toward the chemoattractant or multiple (>1) randomly oriented protrusions. The graph represents the mean ± SEM from three independent experiments (n > 50). *, P < 0.05; **, P < 0.01.