Fig. 4

Knockdown of RNF126 leads to decreased replication stress. A, C Western blot analysis showed AZD6738 (1 μM) led to a greater increase in levels of p-RPA2 and γ-H2AX in parental cells, compared to cells with RNF126 knockdown by RNF126 shRNA#1 (A, MCF7 cells; C, MDA-MB-231). B, D Band intensities were quantified and are presented as bar graphs (Two-way ANOVA, p-RPA2, upper panel; γ-H2AX, down panel). E Schematic of DNA fiber analysis. a, red tracks, IdU; b, green tracks, CldU, scale bar,1 μm. F AZD6738 (1 μM) increased the rate of replication initiation, particularly in cells with intact RNF126, compared with cells depleted of RNF126. The frequency of new origins was calculated as the number of green signals (b) divided by the total of green plus red signals (a + b) (One-way ANOVA, MCF7, left panel; MDA-MB-231, right panel) G AZD6738 induced a greater decrease in replication fork speeds in MCF7 (left panel) and MDA-MB-231 (right panel) cells compared with corresponding cells with RNF126 knockdown. The CIdu/Idu ratio was used to determine elongation (One-way ANOVA). H, I The proportion of cells with foci of CDC45 in MCF7 (H) and MDA-MB-231 (I) cells with or without RNF126 knockdown. Cells were treated with AZD6738 (1 μM) for the indicated times and then subjected to immunofluorescence staining. Representative foci of CDC45 are indicated (Paired t-test, scale bar, 5 μm). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. All presented results are from three independent experiments