(A) Representative images displaying RPA in HU-treated EdU⁺ cells. Scale bars: 20 μm. (B) MCF7 cells were transfected with the indicated siRNA, and 24 hours later, cells were transfected with WT or its mutated CtIP variants. Afterward, cells were pulsed with 10 μM EdU for 20 minutes before addition of 4 mM HU. Cells in S phase (EdU⁺) at the time of HU treatment were Click-iT–labeled (Thermo Fisher Scientific) with an Alexa Fluor 594 azide, and RPA intensity in EdU⁺ cells was enumerated using ImageJ/Fiji. The displayed data represent 3 independent biological replicates, and n ≥ 174 nuclei were analyzed per sample. Holm-corrected multiple testing was performed of ranked data fitted by a linear mixed model, comparing all CtIP variants with WT-CtIP-GFP. ***P < 0.0001. (C) Representative images displaying RAD51 in HU-treated EdU⁺ cells. Scale bars: 20 μm. (D) MCF7 cells were transfected with the indicated siRNA, and 24 hours later, cells were transfected with WT or mutated CtIP variants. Afterward, cells were pulsed with 10 μM EdU for 20 minutes before addition of 4 mM HU. Cells in S phase (EdU⁺) at the time of HU treatment were Click-IT–labeled with an Alexa Fluor 594 azide, and RAD51 foci in EdU⁺ cells were enumerated using ImageJ/Fiji. The displayed data represent 3 independent biological replicates, and n ≥ 207 nuclei were analyzed per sample. Holm-corrected multiple testing was performed of ranked data fitted by a linear mixed model, comparing all CtIP variants with WT-CtIP-GFP.