Figure 4. UVRAG Is Required for Efficient NHEJ Repair.

(A–B) NHEJ assay in HEK293 cells using EJ5-GFP reporter system. HEK293 cells stably expressing the NHEJ reporter were transfected with an empty vector, Flag-UVRAG, control shRNA or UVRAG-specific shRNA before the induction of DSBs by SceI transfection. The NHEJ repair activities as assessed by the reconstituted GFP signal were quantified by FACS. Data shown represent mean ± s.d. for data combined from three independent experiments. The expression levels of UVRAG were analyzed by Western blot in (B).

(C–E) UVRAG overexpression promotes DNA end-joining. 293T cells were sequentially transfected with pTracer-vector, pTracer-UVRAG or its mutant derivatives, and linearized pmCherry DNA. 2 days after transfection, cells were fixed and imaged. GFP is an indicator of transfection, while mCherry is an indicator of precise DNA end-joining. Cells from (C) were analyzed by FACS and the DNA end-joining activity was calculated based on the percentage of mCherry-positive cells in the population of GFP-positive cells in (D). Data shown represent mean ± s.d. for data combined from three independent experiments. (E) Immunoblotting shows UVRAG and its mutants expression. Actin serves as a loading control. Bar, 10 μm.

(F) Colony survival assay of Atg5^+/+ and Atg5^−/− iMEFs expressing control shRNA (control sh) or UVRAG shRNA (UVRAG sh), after an increasing dosage of IR. Values in the graphs represent means ± s.d. of triplicate samples and the experiment was repeated at least twice. See also Figure S3D.

(G) IR-sensitivity of M059J and M059K cells upon UVRAG knockdown. M059J and M059K cells depleted of UVRAG for 72 h were treated with an increasing dosage of IR and colony-forming ability was measured. Values in the graphs represent means ± s.d. of triplicate samples and the experiment was repeated at least twice. See also Figure S3A–B.