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. Author manuscript; available in PMC: 2019 Dec 4.
Published in final edited form as: Mol Cell. 2019 Jan 24;73(5):885–899.e6. doi: 10.1016/j.molcel.2018.12.008

Figure 6. FEN1 loss is synthetic lethal with BRCA1 and BRCA2 deficiency.

Figure 6

(A) Extracts from B2MUT ovarian cells expressing Cas9 and the indicated gRNAs were immunoblotted with the indicated antibodies. (B) Quantification of signal detected Western blotting in (A), normalized to vinculin. (C-D) MCA assays in which the indicated GFP-labeled B2MUT cells and E2-Crimson-labeled B2WT cells were mixed and co-infected with 3 individual gRNAs to FEN1. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to the average of the negative control Grna-expressing cells shown in Supplemental Figure 3A. Error bars reflect the variability of biological triplicates, and the number of asterisks indicates the statistical significance for the corresponding experiment (*=p<0.05,**=p<0.01,***=p<0.001). (E) MCA assay in which colonic GFP-labeled BRCA2 MUT cells and E2-Crimson-labeled BRCA2 WT cells were mixed and co-treated with the indicated doses of a FEN1 inhibitor. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to negative control gRNA-expressing cells. Error bars reflect the variability of biological triplicates, and asterisks indicate statistical significance as described above. (F) Dependency of BRCA1/2 MUT or BRCA1/2 WT cell lines on FEN1, determined from genome-scale CRISPR-Cas9 essentiality screens across 324 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) (Meyers et al., 2017). (G) List of gRNA-resistant ORFs tested for complementation in BRCA2 MUT cells expressing Cas9 and a FEN1 gRNA. (H) Examination of the ability of the ORFs listed in (G) to rescue the growth defect caused by expression of Cas9 and a FEN1 gRNA. BRCA2 MUT ovarian cells were co-infected with lentivirus expressing a FEN1 gRNA and the indicated gRNA-resistant FEN1 ORF or negative control peptide. After selection and growth for 8 d, survival was quantified with a FACS-based cell counting method. (I) Extracts from BRCA2 WT cells expressing Cas9, a validated FEN1 gRNA, and the indicated gRNA-resistant ORF were immunoblotted with the indicated antibodies; all panels are derived from the same blot.