Fig. 2: NR2F1-AS1 (NAS1) promotes metastatic dormancy of breast cancer.

a Expression heatmap of differential lncRNAs in MCF10 cell lines. b Northern blot of NAS1 in CA1h-P2 after NAS1 knockdown, with 18S and 28S ribosomal RNAs as the markers. Northern blotting was repeated three times independently with similar results. c Analyses of total and proliferative GFP⁺ tumor cells seeded in lungs 4 days after intravenous injection of CA1h-P2 with NAS1 knockdown (n = 30 RMFs from 3 mice of each group). The triangles indicate GFP and EdU double-positive cells. d Numbers of pulmonary nodules 4 months after intravenous injection of CA1h-P2 with NAS1 knockdown (n = 7 mice in each group). e IF staining of lung tissue sections in (d). f Analyses of total and proliferative GFP+ tumor cells seeded in lungs 4 days after intravenous injection of CA1h-P1 with NAS1 overexpression (n = 30 RMFs from 3 mice). The triangles indicate GFP and EdU double-positive cells. g Number of pulmonary nodules 4 months after intravenous injection of CA1h-P1 with NAS1 overexpression (n = 8 and 9 mice without or with NAS1 overexpression, respectively). h IF staining of lung tissue sections in (g). i H&E staining (left) and invasion depth (right) of tumor edges after orthotopic injection of NAS1-overexpressing 4175-LM2 cells in NOD/SCID mice (n = 24 and 27 RMFs from 8 and 9 mice, respectively). j BLI quantification (left) and representative images (right) of lung metastasis of the mice in (i) 9 weeks after injection (n = 8 and 9 mice without or with NAS1 overexpression, respectively). k H&E and IF staining of lung tissue sections in (j). H&E and IF staining in e, h, k were performed on lung sections of three mice in each group with similar results, and representative images were shown. Scale bar, 100 μm. Data represent mean ± SD (j), mean ± SEM (c, f, I), or mean with data points (d, g). Statistical significance was determined by a two-tailed unpaired t test (c, d, f, g, i) or two-sided Mann–Whitney test (j).