Fig. 4: RFC4 promotes NICD1 protein stability to form a positive feedback loop.

a, b WB analysis of full-length Notch1, total NICD1, and subcellular distribution of NICD1 in indicated cells. c The effect of silencing RFC4 on the levels of K48-linked polyubiquitination of NICD1 was evaluated by immunoprecipitation of FLAG-tagged NICD1 in A549, H1975, LC1, and LLC cells. A dominant-negative mutant form of HA-tagged ubiquitin (UbK48R-HA) was used as a negative control. d The effect of overexpressing RFC4 on the half-lives of NICD1 in A549 cells and silencing RFC4 on the half-lives of NICD1 in H1975 and LC1 cells treated with cyclohexamide (CHX). e Dual-luciferase assays revealed Notch signaling activities in indicated cells. f GSEA analysis of the TCGA lung cancer datasets shows correlation between RFC4 and Notch signaling up signatures. g Pearson correlation analysis of the RNA-seq data of 971 NSCLC tissues in the TCGA lung cancer datasets showed the correlation between RFC4 expression and HES1, HEY1, HEY2, and NRARP levels. h Representative IHC staining images of two cases for RFC4, NICD1, and HES1 in the same set of consecutive NSCLC tissue slices, and correlations between RFC4 expression and levels of nuclear NICD1 and HES1 in 219 cases of NSCLC specimens are shown. Scale bar: 20 μm. Representative images of three independent reproducible experiments are shown (a–d). Data in panel e, f, h are presented as mean ± SD derived from three independent experiments. Two-way ANOVA multiple comparison analysis (e), two-tailed Student’s t-test (g), and cross-tabulation with two-tailed Chi-square test (h) was used for statistical analysis. Source data are provided as a Source Data file.