Figure 5.

PNUTS is required for NHEJ in Xenopus egg extract and human cells. (A) As described in Materials and Methods, HeLa cells were treated with control (Ctr) or PNUTS siRNA, or with PNUTS siRNA and then reconstituted with siRNA resistant GFP-PNUTS (Add-back). NHEJ assay was performed using a GFP-expressing template linearized with Nhe1, as described in Material and Methods. The repair efficiency, measured by GFP expression, is shown. The cell lysates were analyzed by immunoblotting for PNUTS and β-actin. (B) The chromosomal NHEJ reporter cells were transfected with control or PNUTS siRNA. The cell lysates were analyzed by immunoblotting for PNUTS and β-actin. The chromosomal NHEJ assay was performed as in Figure 2C. The repair efficiency, measured by GFP expression, is shown. A minimum of three experiments were carried out in the above panels, and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired two-tailed Student's t-test.