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. 2020 Aug 1;2(3):zcaa013. doi: 10.1093/narcan/zcaa013

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© The Author(s) 2020. Published by Oxford University Press on behalf of NAR Cancer.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — XRCC1 plays prominent roles in replication stress responses and DSBR in BRCA2-deficient cells. (A) Dependence of EJ2-U2OS MMEJ events on BRCA1 and BRCA2. Cells were treated with siRNA according to (32). Data are plotted relative to ctrl siRNA-treated samples and scaled according to their fold depletion as measured via western blot. (B) Western blot of inducible knockdown of BRCA2 in scr-U2OS (scr) and BRCA2-U2OS (BRCA2) cells. scr-U2OS and BRCA2-U2OS (also referred to as scr and BRCA2 or B2) cells can be induced with doxycycline to express scrambled shRNA and BRCA2 shRNA, respectively. (C) Clonogenic survival assay of scr-U2OS and BRCA2-U2OS cells treated with ctrl or XRCC1 siRNA and ATRi + HU. (D) Neutral comet assay in scr-U2OS and BRCA2-U2OS cells treated with ctrl and XRCC1 siRNA and/or HU. Cells were treated with 3 mM HU for 8 h where indicated. At least 50 cells were analyzed for each experiment. (E) Relative fraction of scr-U2OS and BRCA2-U2OS cells positive for the DSB marker γH2AX by immunofluorescence. XRCC1 or ctrl siRNA-treated cells were treated with 3 mM HU for 3 h and then allowed to recover for the indicated times. Cells were marked positive if they contained >10 foci. The number of cells analyzed in each experiment ranged from 87 to 158. P-values were obtained from pairwise z-tests between control and each of the other conditions. *P-value 0.034, P(α)_0.05 0.565; **P-value 0.006, P(α)_0.05 0.793. (F) MTT assay of scr-U2OS and BRCA2-U2OS treated with either ctrl siRNA or XRCC1 siRNA and exposed to varying concentrations of the PARP inhibitor rucaparib for 120 h.