Figure 4
From: MED1, a novel binding partner of BRCA1, regulates homologous recombination and R-loop processing
Effects of MED1 on DNA damage kinetics. (a) Comet assay with U2OS cells. U2OS cells were seeded onto 6-well plates and transfected with siRNA for 24 h. Cells were then irradiated with 2 Gy of IR light for 10 min. After 24 h, cells were collected using the CometAssay Kit (4250-050-K, Trevigen). Samples were processed, and nucleic acids were stained with SYBR Gold and analyzed by confocal microscopy. More than 100 comets were analyzed per sample, where the tail moment was calculated as %DNA in tail length, and DNA damage was prolonged in MED1-deficient cells 24 h after IR irradiation. Unpaired t test, n = 100, **p < 0.001, ***p < 0.0001. (b) Western blotting of U2OS cells. U2OS cells were seeded, subjected to siRNA transfection, irradiated with 2 Gy of IR, and harvested at 0–1–2–3–6–12–24 h after irradiation. Proteins were purified from whole cell extracts and analyzed by western blotting. Prolonged and delayed phosphorylation of ATM and Chk2 was observed in MED1-deficient cells. siCtrl MISSION siRNA Universal Negative Control (Sigma Aldrich).
