Figure 1
SCN5A/NaV1.5 expression and activity promotes the acquisition of a mesenchymal phenotype in MDA-MB-231 breast cancer cells. (A), Representative scanning electron microscopy micrographs performed from MDA-MB-231 shCTL and shNaV1.5 cells. Scale bar, 10 µm. As shown as an illustration underneath the micrographs, MDA-MB-231 shCTL cells (left panel) show fast NaV1.5-related inward current, with a small persistent component, when recorded with a voltage step from −100 mV to −5 mV, while MDA-MB-231 shNaV1.5 cells (right panel), knocked-down for the expression of the SCN5A gene, do not. (B) A cell circularity index was calculated from SEM micrographs taken from shCTL and shNaV1.5 cells (n = 40 and 67 cells per condition, respectively). P = 0.003 (Mann-Whitney rank sum test). (C) Number of filipods per cell counted from SEM micrographs taken from shCTL and shNaV1.5 (n = 24 and 29 cells per condition, respectively). P = 0.002 (Mann-Whitney rank sum test). (D) Summary of cancer cell invasiveness results from 3 independent experiments, for MDA-MB-231 shCTL and shNaV1.5 cells. Results indicate the total number of cells counted per field taken at the x20 objective. Five fields were counted for each individual Matrigel-coated invasion insert, n = 15 inserts (5 independent experiments). P = 0.0013 (Mann-Whitney rank sum test). (E) mRNA expression levels of SCN5A, SNAI1, ZEB1, ETS1 and TWIST1 genes assessed by RT-qPCR in MDA-MB-231 shNaV1.5 cells and expressed as ratios to expression levels in MDA-MB-231 shCTL cells (n = 3–8 separate experiments). SCN5A and SNAI1 were significantly lower in shNaV1.5 compared to shCTL cells (p < 0.001, Mann-Whitney rank sum test). (F) mRNA expression levels of SCN5A, SNAI1 and ZEB1 genes assessed by RT-qPCR in MDA-MB-231 shCTL cells treated with either tetrodotoxin (TTX, 30 µM) or veratridine (Vera, 50 µM) and expressed as ratios to control condition (vehicle). (n = 5–8 separate experiments). TTX treatment significantly reduced the expression level of SNAI1 gene (P = 0.033, Mann-Whitney rank sum test) while Vera increased it (P = 0.003, Mann-Whitney rank sum test).
