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. 2011 Jun 7;39(17):7465–7476. doi: 10.1093/nar/gkr454

Figure 5.

Figure 5.

Dek-deficient MEFs exhibit increased levels of DNA strand breaks and decreased repair by NHEJ. (A) Primary MEFs were isolated from Dek wild-type, heterozygous, and knockout MEFs and treated with 1 mM HU for 24 h. Cells were then collected, suspended in agarose gel, lysed and subjected to electrophoresis. Cells were stained with SYBR green and assessed for DNA damage by determining the tail moment. Columns, mean value of the tail moment from three independent experiments; bars, SEM. (*P < 0.05, ***P < 0.001). (B) Primary MEFs were subjected to hydroxyurea (HU), camptothecin (C) and etoposide (E) treatment for 3 h, and protein lysates harvested and analyzed by western blot analysis for DEK, γH2AX and actin. To confirm the slight baseline increase of γH2AX in the knockout MEFs (compare lanes 1, 5 and 9), untreated samples were rerun separately (right panels). (C) Dek knockout MEFs were retrovirally infected with an empty vector (R780) or a DEK-expressing vector (R780-DEK). Following flow cytometric sorting, the cells were treated and analyzed as in (B). (D) Wild-type and Dek knockout MEFs were co-transfected with pEGFP-Pem1-Ad2 linearized with I-SceI along with DsRed-Express to control for transfection efficiency. The cells were harvested after 3 days and analyzed by flow cytometric analysis. DSB repair frequency was calculated from the percentage of EGFP positive cells divided by the percentage of transfection efficiency. DSB repair frequency of Dek wild-type MEFs were set to 100% each (absolute mean value: 80 × 10−2). Columns, mean value of four independent experiments; bars, SEM. (**P = 0.01) (E) Wild-type and Dek knockout MEFs were transfected with a repair reporter plasmid to detect NHEJ along with an I-SceI expressing vector. One day post-transfection, cells were collected and analyzed for EGFP expression by flow cytometry to determine repair efficiency. DSB repair frequency was calculated from the percentage of EGFP positive cells divided by the percentage of transfection efficiency in each case. DSB repair frequency of Dek wild-type MEFs were set to 100% each (absolute mean value: 30.0 × 10−3). Columns, mean values of 10 measurements per cell type and construct; bars, SEM (*P < 0.05).