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. 2017 Jun 13;7:3332. doi: 10.1038/s41598-017-03211-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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TRADD is required for non-homologous end-joining repair. (a) Knockdown efficacy for TRADD in DNA repair reporter cell lines EJ5 and DR. (b) After 48 hours transfection with TRADD, RPA80, or BRCA1 targeting siRNAs into reporter cell lines, each siRNA was again cotransfected with an I-SceI endonuclease construct. After 72 hours, GFP positive cells were analyzed with a flow cytometer (FACScan). **P < 0.01; ***P < 0.001; n.s., not significant (ANOVA). (c,d) After 1 hour with laser microirradiation, endogenous NHEJ repair factors were stained with each antibody at DNA break sites: 53BP1 (c); Ku70/80 and 53BP1 (d). Scale bars, 10 μm. (e,f) Endogenous HR repair factors were stained as described in c. RAD51 (e); RPA32 and RAD51 (f). γH2AX was used as a DNA damage marker at DNA break sites in (c) and (e). Scale bars, 10 μm. (g) The protein levels of repair factors in TRADD depletion. EJ-5 or DR cells were transfected with TRADD siRNAs (#1 or #2) or control siRNA. The levels of repair factors were detected by using target antibody, respectively. Total protein levels were verified with Ponceus S staining and tubulin antibody as a loading control.