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. 2017 Nov 8;7:15031. doi: 10.1038/s41598-017-15172-4

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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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Depletion of FANCD2 plus MK-8776 treatment in SK-MK-1 cells led to marked accumulation of DNA damage induced by gemcitabine. (A) Before and after transfection with siFANCD2, SK-MES-1 cells were treated with gemcitabine or MK-8776 alone, or gemcitabine plus MK-8776, culture in fresh medium for another 48 h, fixed and immunostained with an anti-γH2AX antibody. (B) The percentage of γH2AX foci positive cells was quantified using Matafer Software (siFANCD2 plus MK-8776 as compared with siControl, *P < 0.001; compared with siFANCD2 or MK-8776, *P < 0.05; siFANCD2 and MK-8776 as compared with siControl, ^# P < 0.01). (C,D) Before and after transfection with siFANCD2, cells were treated with methods as described above, and fixed and immunostained with RAD51 antibody. The percentage of cells with >10 RAD51 foci was quantified from Image Software (siFANCD2 plus MK-8776 as compared with siControl, *P < 0.001; compared with siFANCD2 or MK-8776, *P < 0.01). (E,F) Before and after transfection with siFANCD2, SK-MES-1 cells were treated with gemcitabine or MK-8776 alone, or gemcitabine plus MK-8776. Alkaline comet assay was used to determine SSBs and DSBs, and the images show detectable comet tail when visualized under a fluorescent microscope. Tail moment in the cells were quantified using Comet Score Software version 1.5 (siFANCD2 plus MK-8776 as compared with siControl, *P < 0.001; compared with siFANCD2 or MK-8776, *P < 0.05; siFANCD2 and MK-8776 as compared with siControl, ^# P < 0.01).