Figure 2

Ranolazine inhibits the Na _V 1.5-mediated breast cancer cell invasiveness in vitro. A, SCN5A mRNA expression assessed by real-time qPCR in shCTL and shNa_V1.5 cells (n = 10 separate experiments) and compared with a Mann–Whitney test. B, Mean ± s.e.m. peak INa recorded in 23 shCTL cells and in 20 shNa_V1.5 cells under a depolarization from −100 to −5 mV (Mann–Whitney test). Representative currents are shown underneath. C, shCTL and shNa_V1.5 cell growth and viability after 5 days, expressed relative to the shCTL cell line (n = 3 independent experiments). D, Cell viability of shCTL after 5 days of growth in presence of increasing concentrations of ranolazine, from 0.1 to 100 μM, and expressed relative to the control condition without ranolazine (vehicle). E, Effect of 30 μM tetrodotoxin (TTX) or 50 μM ranolazine (Rano) on shCTL and shNa_V1.5 human breast cancer cell invasiveness (Kruskal-wallis analysis followed by a Dunn’s test). F, shCTL and shNa_V1.5 cells were cultured for 24 h on a Matrigel™-composed matrix treated with 50 μM ranolazine (Rano) or not. F-actin cytoskeleton was stained with phalloidin-AlexaFluor594. A cell circularity index was calculated using ImageJ© software (n = 138–238 cells analysed, Mann–Whitney test). G, shCTL cells were cultured on a Matrigel™-composed matrix containing DQ-Gelatin® for 24 h in presence or not of 50 μM ranolazine. A “Matrix-Focalized-degradation activity index” was calculated as being the number of pixels corresponding to the co-localization of F-actin condensation areas (F-actin cytoskeleton was stained with phalloidin-Alexa594) and focal spots of DQ-gelatin proteolysis (coloc) (7). Results are expressed relative to the control condition (CTL, N = 534 cells) without ranolazine (Rano, N = 375 cells) and compared using Mann–Whitney test. Representative pictures are shown on the left. Statistical significance is indicated as: *p <0.05; **p < 0.01 and ***p < 0.001. NS stands for not statistically different.