FIG. 5.

Accumulation of Rad51-knockdown mESCs in G2/M and its effect on DNA damage checkpoint activation. (A) Analysis of cell cycle profiles after Rad51 knockdown in mESCs and MEFs. After siRNA transfection for 48 h, cells were harvested and stained with PI for FACS analysis as described in “Materials and Methods” (As; asynchronous cells). Data were quantified using FlowJo software (bottom). Error bars indicate mean±SD. (B) mESCs transfected with siNS or siRad51 were attempted to synchronize at G1/S phase using double thymidine. The cell cycle profiles of the cells were then analyzed using FACS Calibur. The population of each cell cycle phase was quantified with FlowJo software (middle). The level of Rad51 was determined by immunoblot analysis (bottom). (C) Activation of the DNA damage checkpoint by depletion of Rad51. Cells were harvested 48 h after siRNA transfection, and markers involved in cell cycle control and DNA damage checkpoint signaling were detected by immunoblot analysis. (D) The relative proportion of cells at indicated cell cycle phase after Rad51 siRNA transfection in the presence of mirin.