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. 2022 Jun 27;13:3662. doi: 10.1038/s41467-022-31365-6

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© The Author(s) 2022

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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Fig. 6 — a Shown is the LMNA-HDR reporter (not to scale) involving a Cas9/sgRNA that induces a DSB in LMNA exon 1, and a plasmid donor such that HDR causes mRuby2 to be expressed from the LMNA locus. The mRuby2 frequencies are normalized to transfection efficiency with parallel GFP transfections. b BRCA2 is required for HDR by the LMNA-HDR assay, but has modest effects on No Indel EJ. Cells were transfected with siRNA that is non-targeting (siCtrl) or targets BRCA2 (siBRCA2). n = 6 biologically independent transfections. Statistics with unpaired two-tailed t test using Holm–Sidak correction. ****P < 0.0001, **P = 0.00305. Immunoblot shows levels of BRCA2. c DNAPKcs, XLF, and XRCC4 suppress HDR to similar degrees. n = 6 biologically independent transfections. Statistics as in (b). ****P < 0.0001, ***P = 0.00069, **P = 0.00117, n.s. not significant. d Suppression of HDR. n = 6 biologically independent transfections. Statistics as in (b). ****P < 0.0001, Parental EV DMSO vs. 1000 nM M3814 ***P = 0.000493, XLF-KO XLF-WT DMSO vs. XLF-K160D DMSO ***P = 0.000404, XLF-KO EV DMSO vs. 1000 nM M3814 **P = 0.0203, XLF-KO XLF-WT 250 nM M3814 vs XLF-K160D 250 nM M3814 *P = 0.0249, ^†P = 0.0386 but not significant after correction (unadjusted P-value), n.s. not significant. e Suppression of SVs. Shown is the GFPd2-SV reporter (not to scale) that is chromosomally integrated to an FRT site in HEK293 cells. Stable expression of Cas9 and various sgRNAs (DSB-H, DSB-G, and DSB-L) can induce GFP-negative cells (GFP-neg). n = 6. Statistics as in (b). ****P < 0.0001, **P = 0.00136, n.s. not significant. f IR sensitivity. HEK293 stable cell lines were treated with DMSO or M3814, and 0 Gy or 1 Gy IR dose, and plated to form colonies. Fraction clonogenic survival was determined relative to DMSO 0 Gy for each cell line. n = 6 biologically independent wells of seeded cells. Statistics as in (b). ****P < 0.0001, n.s.= not significant. Error bars = SD. Data are represented as mean values ± SD.