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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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Depletions of the FA pathway factors sensitized LSC cells to MK-8776. (A,B) The viability analysis were performed using the CCK-8 assay in SK-MES-1 and KLN205 cell lines depleted of the FA pathway factors after MK-8776 treatment for 6 h. (siControl as compared to siFANCL, siFANCD2 and siBRCA2, *P < 0.05; ^# P < 0.01). (C,D) The two LSC cell lines were treated with MK-8776 following transfection with various siRNA as indicated. The cells were stained by crystal violet and total colonies were counted after two weeks. Colony numbers of control-treated cells were set as 100%. (siControl as compared to siFANCL, siFANCD2 and siBRCA2, ^* P < 0.01; ^# P < 0.005). (E) Western blotting detecting monoubiquitination of FANCD2 and phosphorylation of Cdc25C (S216) and CHK1 (S317) in SK-MES-1 cells transfected with siControl, siFANCD2 and siBRCA2. The cells were either treated with MK-8776 (0.5 μM+ , 1 μM++) or DMSO control for 6 h. GAPDH was used as loading control. Uncropped images are presented in Supplementary Figure S6. (F) SK-MES-1 cells were treated with MK-8776 at the indicated doses for 6 h following transfection with siFANCL or siBRCA2, and fixed and immunostained using anti-FANCD2 antibody. (G) The percentage of cells with >20 FANCD2 foci was quantified using Spot Advanced RT software (the cells treated with 0.5 μM MK-8776 as compared to the cells treated with 0 μM MK-8776, ^* P < 0.05, ^# P < 0.01; the cells treated with 1 μM MK-8776 compared to the cells treated with 0 μM MK-8776, ^** P < 0.01, ^## P < 0.001). (H and I) SK-MES-1 cells were treated with 5 nM gemcitabine for 4 hours. The drug was then replaced with MK-8776 or CHK2 inhibitor II at the indicated doses and the cells harvested after an additional 6 hours. Cell lysates were analyzed by Western blotting for the indicated protein.