Fig. 4: NAS1 suppresses breast cancer lung metastasis by NR2F1.

a NR2F1 expression in MCF10CA1a, MCF10CA1h, P1, and P2, or after NAS1 knockdown or overexpression. Relative quantitation of NR2F1 expression after NAS1 knockdown was shown for CA1h-P2 cells. b Expression of EMT markers after NR2F1 overexpression in MCF10AT. c Tumorsphere formation of MCF7 and MCF10AT after NR2F1 overexpression (n = 3 culturing experiments). d–i Analyses of CA1h-P2 cells with NAS1 knockdown and/or NR2F1 overexpression. Shown are protein levels of EMT markers (d), flow cytometric analyses of the ALDH⁺ BCSCs (e), tumorsphere formation (f, n = 3 culturing experiments), representative IF images (left), and quantitation of EdU⁺ tumor cells (right) in lungs 4 days after intravenous injection of the cells (g, n = 30 RMFs from 3 mice), lung surface nodules 18 weeks after intravenous injection (h, n = 7, 8, 8 and 8 mice, respectively), and the lung metastasis incidence (i). The triangles indicate metastatic nodules on the lung surface (h). Scale bar, 100 μm. Data represent mean ± SD (c, f), mean ± SEM (g) or mean with single points (h). Arrows point to tumor nodules in (h). Statistical significance was determined by a two-tailed unpaired t test. Experiments in a, b, d, e were repeated at least three times independently with similar results; data from one representative experiment are shown.