(a) The expression of SCN4B gene was studied by RT–qPCR in human mammary epithelial non-cancer MCF-10A and cancer MCF7, MDA-MB-468, MDA-MB-435s and MDA-MB-231 cell lines. Results are expressed as relative to that of HPRT-1 gene (n=7–12) and presented as mean values±s.e.m. *, significantly different from MCF-10A at P<0.05 (MW). (b) The expression of SCN4B/β4 protein was studied by densitometric analysis of western blot experiments in same cells as in a. Results are given as the amount of SCN4B/β4 protein relative to that of HSC70 (n=5) and presented as mean values±s.e.m. *, significantly different from MCF-10A at P<0.05 (MW). The image on top shows a representative western blotting experiment. (c) The expression of SCNxB genes was analysed in MDA-MB-231-Luc cells by reverse transcription–PCR. Plasmids encoding human SCNxB genes were used as positive controls for PCR primers. (d) Representative western blotting experiments showing protein expression for β1 (SCN1B), β2 (SCN2B) and β4 (SCN4B) in MDA-MB-231-Luc cells. (e) Cells were transfected with scrambled siRNA (siCTL) or with siRNA directed against the expression of the SCN1B gene (siSCN1B), the SCN2B gene (siSCN2B) or the SCN4B gene (siSCN4B). The efficacy of siRNA transfection was assessed by western blotting experiments 48 h after transfection. HSC70 was used as a control for sample loading. (f) Representative images of fixed and haematoxylin-stained MDA-MB-231-Luc cells on invasion inserts. Cancer cells were transfected with scrambled siCTL or with specific siRNA. Scale bars, 50 μm. (g) Summary of cancer cell invasiveness results (n=8) for MDA-MB-231-Luc cells transfected with siCTL or siSCNxB. Results were expressed relative to siCTL and presented as mean values±s.e.m. ***, statistically different from siCTL at P<0.001 (Student’s t-test). (h) Representative image of a zebrafish embryo injected in the yolk sac with MDA-MB-231-Luc cells stained with CM-Dil and showing sites of colonization. Scale bars, 500 μm. Below is a magnification of the highlighted region containing human cancer cells (see arrows) colonizing organs of the embryo. (i) Zebrafish colonization index of siCTL or siSCN4B cells. Numbers in brackets indicate the number of embryos examined for each condition, from three different experiments. Results are presented as mean values±s.e.m. **, statistically different from siCTL at P<0.01 (Student’s t-test).