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. 2021 Jun 29;10(7):1624. doi: 10.3390/cells10071624

Figure 1.

Figure 1

NaVβ4 downregulation induces morphological changes in non-cancer mammary cells. (a) The expression level of the SCN4B gene, coding for NaVβ4, was analysed from datasets from The Cancer Genome Atlas (http://cancergenome.nih.gov, accessed on 19 December 2020), from the US National Cancer Institute, in the non-tumoral adjacent tissue (n = 178), and in the different stages of primary breast tumours: I (n = 125), IIA (n = 243), IIB (n = 115), IIIA (n = 85), IIIB (n = 10), IIIC (n = 31), IV (n = 4). For each array, data were log2-transformed and centred to the median. ***, statistically different with p < 0.001 (Mann–Whitney rank sum test) when comparing with adjacent non-tumoral tissue; *, p < 0.05 when comparing Stage I with stage IIA. (b) NaVβ4 protein expression level was assessed by western blotting in non-cancer MCF10A human mammary epithelial cells and in human breast cancer MDA-MB-231 cells. The upper section shows a WB representative of 5 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of NaVβ4 protein expression in the two cell lines expressed relatively to that of MCF10A. *, statistically different with p < 0.05 (Mann–Whitney rank sum test). (c) NaVβ4 protein expression level was assessed by western blotting in control MCF10A cells and in cells stably knocked down for the expression of SCN4B gene (MCF10A Crβ4). The upper section shows a WB representative of 8 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of NaVβ4 protein expression in the two cell lines expressed relatively to that of MCF10A CTL (n = 8). *, statistically different with p < 0.05 (Mann–Whitney rank sum test). (d) Representative images of MCF10A CTL and MCF10A Crβ4 cells in phase contrast microscopy. Scale bar, 50 µm. (e) Maximal cell length (n = 31 MCF10A CTL and n = 20 MCF10A Crβ4) and, in (f), number of intercellular contacts per cell (n = 60 MCF10A CTL and n = 57 MCF10A Crβ4), assessed from images taken as in (d). Cells were randomly selected from pictures, and the number of joint cells was manually counted. ***, statistically different with p < 0.001 (Student’s t-test). (g) MCF10A CTL and MCF10A Crβ4 cells were stained for the identification of nuclei (DAPI, blue staining) and F-actin (phalloidin-594, red staining). Scale bar, 125 µm. (h) Mean cell area (n = 40 MCF10A CTL and n = 40 MCF10A Crβ4) and, in (i), F-actin fluorescence intensity per cell surface (n = 100 MCF10A CTL and n = 100 MCF10A Crβ4) were calculated from images taken as in (g). ***, statistically different with p < 0.001 (Student’s t-test).