Fig. 2: NELF depletion leads to reduced stemness-like traits and downregulated EMT pathway.

a MA plot showing gene expression changes between NELF-E KO and WT SUM159 cells. Red and blue points indicate significantly upregulated (n = 388) and downregulated (n = 540) genes, respectively. b GSEA enrichment plot for stemness and epithelial-mesenchymal transition pathways in NELF-E KO SUM159 cells. c Western blot analysis of NELF-E and NELF-A in WT, NELF-E KO, and NELF-E rescue SUM159 cells. β-actin was used as the loading control. d Quantification of wound healing assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells. Scale bar = 100 μm. e Quantification of mammosphere formation assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells (n = 4). Scale bar = 100 μm. f Quantification of invasion assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells (n = 3). Scale bar = 100 μm. g Body weight (mean ± SEM) of mice injected with 1.25 × 10⁶ WT and NELF-E KO SUM159 cells, respectively (n = 8, each group). h Lungs inflated and fixed with 10% neutral buffer formalin from mice in WT (n = 8) and NELF-E KO group (n = 8). i Representative images of H&E staining in lung tissues at three magnifications (mf: metastatic foci). From top to bottom, Bar = 2 mm, 500 μm, and 100 μm, respectively. j Quantitative analysis of lung metastasis presented as % of occupation by metastasis and the number of metastatic sites per lung (n = 8/group). k Graph showing quantification of mammospheres in WT, NELF-E KO, and NELF-E rescue MCF7 cells at primary (n = 3), secondary (n = 4), and tertiary passages (n = 4). l MCF7 cells from tertiary spheroids were seeded at a density of 1000 cells per well and incubated for 2 weeks. The colonies were then fixed and stained with crystal violet. m Flow cytometry analysis and quantification of the CD24^low/CD44^high population in MCF7 tertiary mammospheres (n = 3). Blots and images are representative of at least three independent experiments. Data in (d, e, f, j, k, m) are presented as mean ± SD. p-values are determined by a two-tailed Student’s t-test. Source data are provided as a Source Data file.