Fig. 4

Endonuclease/exonuclease/phosphatase family domain-containing-1 (EEPD1) depletion rescues stressed replication forks in radiation repair protein 52 (RAD52)-depleted breast cancer susceptibility protein-1 (BRCA1)-deficient breast cancer cells. DNA fiber analysis of stalled, restarted or new forks in MDA-MB-436 BRCA1^-/- cells transfected with control, EEPD1 and/or RAD52 siRNA. a Schematic diagram depicts steps for the DNA fiber assay and representative images of DNA fibers from each condition. Forks prior to stress with hydroxyurea (HU) are stained red with iodo-deoxyuridine (IdU), new forks after relieving stress are stained green with chloro-deoxyuridine (CldU). Quantitative analysis of stalled (only red fibers) (b), restarted (red then green fibers) (c) and new forks (only green fibers) (d) after release from HU replication stress. e, f Quantitative analysis of stalled versus restarted replication forks (IdU:CIdU) ratios under unstressed condition or stressed condition (5 mM HU for 120 min). Three distinct experiments per condition (>121 fibers measured per condition for each experiment)