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. 2018 Dec 21;47(5):2402–2424. doi: 10.1093/nar/gky1279

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — LIG1-depleted cells maintain an intact DNA damage response and suppressed mitotic slippage. (A) Flow cytometry detection of the phosphorylated (Ser139) histone variant H2AX (γH2AX; red overlays) as a surrogate marker for DSBs in G2-arrested and 4-OHT-treated L1^−/fx:Cre cells transfected with 17p TLN or 0 DNA control over 24 h. The top left insert shows a histogram of γH2AX-expressing (8% population; blue overlay) unsynchronized and untreated L1^−/fx:Cre cells exposed to 2 mM hydroxyurea (HU) for 1 h at 37°C. The bottom left insert reveals the basal 1.6% γH2AX-expressing asynchronous and untransfected WT HCT116 cells. The right-hand bar chart shows mean proportions (with SD) of γH2AX-expressing G2-arrested L1^−/fx:Cre cells 24 h post-nucleofection (two replicas). The proportions of cells with defined DNA content in three independent experiments as assessed by DAPI DNA staining of fixed G2-arrested WT, L3^−/−:LIG4^−/− and L1^−/fx:Cre cells (B) pre-nucleofection or (C) 24 h post-nucleofection (pooled 17p TLN and 0 DNA transfections) with detection by image cytometry. (i) Data for each fraction of pre- and post-nucleofection samples is plotted as the mean with standard deviation and assessed using one-way Kruskal–Wallis ANOVA of individual population fractions (significance set as alpha = 0.05) recorded against the colour key code. Differences relating specifically to the 4-OHT-mediated deletion of LIG1 from L1^−/fx:Cre cells are shown in B(ii) and C(ii). Significance was assessed by one-tailed paired T-tests of common fractions. The significant (Mann–Whitney U-tests, 0.05; *, 0.005; **) variations in proportions of WT, L3^−/−:L4^−/− and L1^−/fx:Cre cells treated with 4-OHT are displayed in B(iii) and C(iii).