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. 2020 Jun 1;34(11-12):832–846. doi: 10.1101/gad.336446.120

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© 2020 Rickman et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — Defective ICL repair in BRCA2 DBD mutants results in increased ssDNA that is WRN and DNA2 dependent. (A) Immunofluorescence images of RAD51 foci, 8 h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 focus size. (B) Quantification of RAD51 foci 1 h, 8 h, and 24 h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate SD of two independent experiments (≥200 cells per experiment). (C) Quantification of RAD51 foci 8 h after 12 Gy IR of BJ WT fibroblast, wild-type HSC62 (HSC62^WT) clones 1–3, and HSC62 uncorrected patient cell line (HSC62^mut). (D) Quantification of RAD51 foci 24 h following 1-h treatment with 3 µM MMC. Error bars indicate SD of three independent experiments (≥200 cells per experiment). (E) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1 h, 8 h, and 24 h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2^WT), BRCA2^8488-1G>A BJ clones 2–3, BRCA2^8524C>T BJ clones 1–2, and a BRCA2 homozygous truncation mutant, c.8531dupA (BRCA2^Trun). Error bars indicate SD of three independent experiments (≥200 cells per experiment). (F) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8 h after 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 focus size. (G) Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild-type HSC62 clones (HSC62^WT), and HSC62 uncorrected patient cell line (HSC62^mut). (H) Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC in HSC62^mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared with luciferase control (Luc). Error bars indicate SD of four independent experiments. (I) Quantification of RPA foci 24 h following 1 h treatment with 3 μM MMC in HSC62^mut cells depleted of DNA2, WRN, BLM, or codepleted of WRN and BLM by siRNA compared with luciferase control (Luc). Error bars indicate SD of three independent experiments. (J) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24 h after 1-h treatment with 3 μM MMC. BRCA2^WT, BRCA2^8524C>T, and BRCA2^8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. (K,L) MMC cell survival of BJ BRCA2^WT, BRCA2^8488-1G>A, and BRCA2^8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate SD.