Fig. 9

PLK4 and AURKB regulate cell motility through DAAMs. a MDA-MB-231 cells were stimulated overnight with DMEM or ACM in the presence of DMSO, CK666 (ARP2/3 inhibitor) or SMIFH2 (formin inhibitor). b Cells were transfected with control or SPIRE1 (actin nucleator) siRNAs for 72 h and then treated overnight with DMEM or ACM. Cell motility from cells in (a) and (b) was measured as described (****p < 0.0001; n = 3, at least 50 cells were tracked per condition). c Cells were transfected with control, DAAM1 or DAAM2 individual siRNA oligos for 72 h and then stimulated overnight with DMEM or ACM as indicated. Cell motility was measured as described in Methods (*p < 0.05, ***p < 0.001, ****p < 0.0001; n = 3, at least 50 cells were tracked per condition). d, e MDA-MB-231 cells were stimulated with DMEM or ACM in the presence of DMSO or centrinone + AZD as indicated. Cells were stained for actin and (d) DAAM1, or (e) DAAM2. Bar = 20 µm. Right side box and whiskers plots show the quantification of average signal intensity at the cell cortex, which was measured as described in Methods. Light green is DAAM1, teal is DAAM2 (****p < 0.0001; n = 3, at least 60 cells were measured per condition). f, g Cells were transfected with control, DAAM2 or DAAM1 siRNAs for 72 h, and then stimulated overnight with DMEM or ACM as indicated. Cells were stained for actin and (f) DAAM1, or (g) DAAM2. Bar = 20 µm. Right side plots show the average signal intensity at cortical region for DAAM1 (light green) or DAAM2 (teal), which was measured and plotted as described in Methods (*p < 0.05, ****p < 0.0001; n = 3, at least 60 cells were measured per condition). h MDA-MB-231 cells were transfected with control, DAAM1, DAAM2 siRNAs or the indicated combinations for 72 h and then stimulated overnight with DMEM or ACM. Cell motility was measured as described (***p < 0.001, ****p < 0.0001; n = 3, at least 60 cells were measured per condition). i MDA-MB-231 cells were transfected with control, DAAM1 or DAAM2 siRNAs for 72 h and then stimulated overnight with DMEM or ACM in the presence of DMSO or centrinone + AZD. Cell motility was measured as described (***p < 0.001; n = 3, at least 60 cells were tracked per condition). j Model for DAAM1 and DAAM2 localisation switch at cell cortex upon ACM treatment regulated by PLK4 and AURKB. All the data were plotted as box and whiskers. Boxes represent median and 25th to 75th percentiles, whiskers the minimum and maximum values with each individual cell value superimposed. The data were compared with one-way ANOVA Kruskal–Wallis test and post-tested with Dunn’s multiple comparison test. Arrow indicates the control bar used for comparison