Fig. 5

Replication stress in breast cancer cells with a higher level of RNF126 is mediated by CDK2. A MTT assay showed the effect of co-treatment NU2058 (5 μM) and various concentrations AZD6738 on MCF7 (left panel) and MDA-MB-231 (right panel) cell proliferation (Two-way ANOVA). B, C The expression of cleave-PARP in MCF7 (B) and MDA-MB-231 (C) cells with or without RNF126 depletion. Cells treated with AZD6738 alone or combined with NU2058 (5 μM) at indicated time points. The indicated proteins were analyzed by western blot. Band intensities were quantified and are presented as bar graphs (Two-way ANOVA). D, F Western blot analysis showed NU2058 (5 μM) decreased the levels of p-RPA2 and γ-H2AX in parental cells caused by AZD6738 (D, MCF7; F, MDA-MB-231). E, G Band intensities of p-RPA2 (left panel) and γ-H2AX (right panel) were quantified and are presented as bar graphs (Two-way ANOVA). H RNF126 knockdown by shRNAs led to decreased expression of CDK2 protein in MCF7 (left panel) and MDA-MB-231 cells (right panel). I Band intensities were quantified and are presented as bar graphs (Two-way ANOVA, left, MCF7; right, MDA-MB-231). J The expression of an E3 ligase mutant of RNF126 did not affect CDK2 protein expression. MCF7 (left panel) or MDA-MB-231 (right panel) cells were transfected with control vector, RNF126WT, or E3 ligase-deficient RNF126 (RNF126 C229A/C232A) plasmids and levels of CDK1/CDK2/CDK5 proteins were then detected by Western blotting. K Band intensities were quantified and are presented as bar graphs (Two-way ANOVA, left, MCF7; right, MDA-MB-231). L RNF126 and CDK2 mRNA levels in MCF7 (left panel) or MDA-MB-231 (right panel) cells, with or without RNF126 knockdown by shRNAs (One-way ANOVA). M The level of CDK2 mRNA expression in MCF7 (left panel) or MDA-MB-231 (right panel) cells, with RNF126WT/RNF126 C229A/C232A overexpression (One-way ANOVA). N The CDK2 mRNA relative expression levels in MCF7 and MDA-MB-231 without RNF126 (not CDK2) expression (Paired t-test). O Cell proliferation assay showed the effects of CDK2 overexpression in MCF7shRNF126#1 (left panel) and MDA-MB-231shRNF126#1 (right panel) cell at indicated time points (Two-way ANOVA). P Model for how RNF126 promotes breast cancer cell proliferation and as a biomarker of ATR inhibition. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. All presented results are from three independent experiments