Figure 6.

E2A–Snail1 axis favors self-renewal and invasiveness of PyMT-NCs. A, Representative immunoblots of the indicated EMT markers and EMT-TFs in NC cell lines stably expressing Snail1 (GFP-Snail1 and CRE-Snail1) and their corresponding controls (GFP-Ø and CRE-Ø). GAPDH was used as a loading control. (s.e., short exposure; l.e., long exposure). B and C, Mammospheres formed by control and Snail1-overexpressing NC cell lines. Representative microphotographs (B) and relative mammosphere forming efficiency (C) at first (1^stG) and third (3^rdG) generations of the indicated NC cells. Values in C are expressed relative to those corresponding to 1^stG in control GFP-Ø cells. Data represent the mean ± SEM of four independent experiments. D, Limiting dilution transplantation experiments of single-cell suspensions obtained after dissociation of 1^stG mammospheres formed by the indicated NC cell lines (n = 3 different cell suspension/condition). TIC frequency and associated probability (left) were calculated with ELDA software (CI, 95%). Representative hematoxylin and eosin images of tumors generated after transplantation of control and Snail1-overexpressing NCs (right). Scale bar, 100 μm. E, Invasion assays on Matrigel chambers of the indicated NC cell lines. Results, expressed in percentage relative to values obtained for NC-GFP-Ø cells, represent the mean ± SEM of three independent experiments. F–H, Schematics of the experimental procedure and hematoxylin and eosin representative lung images (F), number per mouse (G), and number per size category (H) of lung metastatic foci generated by the indicated cell lines upon tail vein injection. Scale bar in F, 2 mm. Midlines in G and barplots in H show the mean values; error bars, SEM; n = 12 mice per condition. P value was calculated by one-way ANOVA test in C, E, and G. ns, not significant.