Fig. 1: RFC4 is essential for Notch activation-induced metastasis and stemness of NSCLC.

a GSEA analysis of gene expression profiles of NSCLC patients with or without lymphatic node (LN) metastasis in the TCGA lung cancer datasets by Kolmogorov–Smirnov test. b IHC determined NICD1 expression in primary tumors of our cohort of 219 NSCLC patients with or without LN metastasis. Two representative cases are shown. Scale bar: 20 μm. c Kaplan–Meier analysis of LN metastasis-free survival of our 219 NSCLC patients by log-rank test, who were divided into low- or high- subgroups according to the median of NICD1 expression. d, e A549-luci-Vector or A549-luci-NICD1 cells were injected via cardiac ventricle into nude mice (n = 5 per group). Representative bioluminescent images of systemic metastasis and ex vivo organ metastases are shown (d), H&E histologically confirmed tumor cells in bone and brain tissue (e). Two representative cases are shown. Scale bar: 100 μm. f, g Nude mice were intravenously injected with A549-luci-Vector or A549-luci-NICD1 cells (n = 5 per group). Representative bioluminescent images (f), picric acid staining, H&E staining, and the numbers of metastatic foci of lung tissue are shown (g). Two representative cases are shown. Scale bar: 100 μm. h Growth curves of tumor xenografts of the indicated cells subcutaneously implanted with different cell numbers and tumor formation frequencies are shown. i Gene expression array analysis showed the most upregulated 10 genes induced by NICD1 overexpression. j The effect of silencing RFC4 on expression of the indicated mRNAs in NICD1-overexpressing cells. k, l The abilities of overexpressing NICD1 or together with RFC4-silenced A549 cells colonize in the lung or to generate tumor xenografts (n = 5 per group). Three representative cases are shown. Scale bar: 100 μm. Error bars represent the means ± SD derived from three independent experiments. Statistical analyses were performed by two-tailed unpaired Student’s t-test (g), two-way ANOVA multiple comparison analysis (h, j–l). Source data are provided as a Source Data file.