Figure 6. Skp2 Regulates HR Repair and DNA Damage Response.
(A) DR-GFP-integrated U2OS cells silenced with control or Skp2 were transfected with mock or I-Scel along with MSCV-GFP as a transfection efficiency control. Two days after IR treatment, cells were collected to analyze HR-repaired GFP-positive cells by flow cytometry assay. Results are presented as mean values ±SD, n = 3.
(B) DR-GFP-integrated U2OS cells silenced with control or Skp2 were transfected with mock or I-Scel. Forty-eight hours after transfection, genomic DNA was extracted for PCR amplification, followed by I-SceI or I-SceI plus BcgI digestion, and PCR products were subjected to gel electrophoresis. This result indicates that Skp2 knockdown impairs HR repair, but not NHEJ repair.
(C) WT and Skp2–/– MEFs were treated with or without IR (6 Gy). Seven hours after IR treatment, cells were harvested for apoptosis assay. Results are presented as mean values ±SD. *p < 0.05, using Student’s t test, n = 3.
(D) PC3 cells silenced with control or Skp2 were treated with various doses of IR, and the survival rate of these cells was determined by colony formation assay. Results are presented as mean values ±SD. *p < 0.05, using Student’s t test, n = 3.