Fig. 1: Nanog interacts with Rad51 and promotes γH2AX accumulation in mouse ES cells.

a Co-IP assay was used to detect the association of Nanog with Rad51. Mouse ES cell extracts were subjected to co-IP with the antibody against Nanog or Rad51, followed by western blotting with the antibodies against Rad51 and Nanog, respectively. Whole cell extract for IP was used as the input control. b Pull-down assay was performed to analyze the direct association of Nanog with Rad51. Bacterially purified GST-tagged Nanog was conjugated to glutathione-sepharose beads, and subsequently incubated with purified His-tagged Rad51. The elution was analyzed by western blot using the anti-Rad51 antibody. GST was used as a control. c Nanog overexpression retarded γH2AX removal. Mouse ES cells expressing HA-tagged Nanog were treated with CPT for 6 h. The medium was changed to fresh CPT-free medium and allowed cells to grow for additional 4 and 8 h, respectively. Whole cell proteins and histones were extracted and subjected to SDS-PAGE. The cells transfected with the mock vector were used as controls. d Representative cell images of the comet assay. Similar CPT treatment and release strategies with c were performed for the Nanog-overexpressed mouse ES cells and mock control cells. e The average percentages of DNA in tails were calculated. The data are based on three independent repeats, and presented as mean ± SEM. *p < 0.05 (Student’s t-test).