Fig. 4

Dishevelled 2 controls ACM-induced cancer cell motility by binding to PLK4, AURKB and CEP192. a Network graph for selected protein interactions identified from a LUMIER screen testing CEP192, PLK4 and AURKs against a collection of 3 Flag-tagged WNT-PCP and centrosomal factors. Edge width reflects the normalised LUMIER intensity ratio that indicates interaction strength (n = 2). b HEK293T cells were co-transfected with 3Flag-CEP192 and 3HA-PLK4 (positive control) or 3HA-DVL2 as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody and 3HA-DVL2 or 3HA-PLK4 were detected by western blotting. Red arrowhead indicates band corresponding to 3HA-PLK4, blue arrowheads indicate 3HA-DVL2 bands (non-phosphorylated and phosphorylated forms), red star on the left side indicates non-specific band (n = 3). c, d GST, GST-AURKB (c) and GST-PLK4 (d) were bacterially expressed, purified and incubated with or without cell lysates from MDA-MB-231 cells. GST pull down was performed and endogenous DVL2 was detected by western blotting. (n = 4 for each). e MDA-MB-231 cells were treated with control or DVL2 siRNAs for 72 h and then stimulated with DMEM or ACM overnight. Cell motility was measured as described in Methods and analysed with one-way ANOVA Kruskal–Wallis test and post-tested with Dunn’s multiple comparison test. Arrow indicates the control bar used for comparison (****p < 0.0001; n = 3, at least 40 cells were tracked per condition). f Cells from (e) were stained with actin and α-tubulin. Enlarged box areas of cortical regions are shown as ROIs in lower panels. Bar = 20 µm (n = 3). g MDA-MB-231 cells stably expressing tetracycline inducible C-terminally HA-tagged DVL2-wt, DVL2-N terminus (Δ361–736) or DVL2-C terminus (Δ1–360) were transfected with control or DVL2 siRNA. After 72 h, cells were treated with or without tetracycline in the presence of DMEM or ACM as indicated. Cell motility was measured as described in Methods and analysed by Mann–Whitney U test two-tailed (****p < 0.0001; n = 3, at least 50 cells were tracked per condition). All the data are 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