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. 2020 Aug 27;2(3):zcaa017. doi: 10.1093/narcan/zcaa017

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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 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. — POLQ is involved in distal EJ. (A) Detection of distal EJ and chromosome translocation in human cells. The human bladder carcinoma cell line EDS-7F2 harbors a pEJ5-GFP construct on one chromosome and a pDsRed-I-SceI construct on another chromosome. For the break end-joining assay, the GFP gene and the DsRed genes are initially inactive due to the lack of a promoter but become activated following NHEJ between the I-SceI-induced DSBs. (B) Following infection with a retrovirus expressing I-SceI, the percentage of cells expressing GFP (distal EJ) or DsRed (chromosome translocation) was determined by flow cytometry. The frequencies of GFP+ (C) and DsRed+ (D) cells were determined in EDS-7F2 (7F2) and two POLQ knockout EDS-7F2 clones (F7 and F10). Error bars represent standard deviation of three separate experiments. The differences between 7F2 and POLQ^−/− cells (F7, F10) are statistically significant (P ≤ 0.05, unpaired t-tests). (E) POLQ knockout U2OS cell lines carry the recombination reporter DR-GFP integrated into the genome. SceGFP is a GFP gene that contains an I-SceI endonuclease site within the coding region. Cleavage of the I-SceI site in vivo and repair by HR directed by the downstream iGFP results in GFP+ cells. (F) HR in POLQ^+/+ cells (DR-U2OS) and POLQ^−/− cells (F5, F10, G6) after I-SceI expression. The differences between DR-U2OS and F5, F10 and G6 cells are not statistically significant (P > 0.05, unpaired t-tests). This result was confirmed from four independent experiments.