Additional file 4: Figure S4. of The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

Single-label DNA fiber analysis of stressed replication fork degradation. MDA-MB-436 BRCA1-/- cells were transiently transfected with control, EEPD1 and/or RAD52 siRNA for 48 h and labeled with IdU for 45 min before proceeding to either 0 h or 10 h incubation with 5 mM HU. DNA degradation at stalled nascent replication forks was measured by fiber analysis. a Schematic diagram depicts steps for the DNA fiber assay and representative images of DNA fibers from each condition. IdU stained red (stalled forks). Quantitative analysis of IdU track length frequency at unstressed (no HU) (b), or HU-treated DNA fibers (c) from each condition. d Bar chart from the HU-treated IdU track length frequencies analysis. c and d are the same data. Co-depletion of both RAD52 and EEPD1 restores stressed replication fork degradation back to control levels. Three distinct experiments per condition (>100 fibers measured per condition for each experiment). (PDF 419 kb