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. 2023 Sep 9;116(1):138–148. doi: 10.1093/jnci/djad182

Figure 4.

Figure 4.

BARD1 +/mut cells are deficient in DNA repair and sensitive to poly (ADP-ribose) polymerase (PARP) inhibition. A) Schematic of the Clover-LMNA homology-directed repair assay (39). B) Representative flow cytometry plots of IMR-5 wild-type (WT) and BARD1+/E287fs cells cotransfected with pX330-LMNA1 guide RNA and pCR2.1 Clover-LMNA repair template plasmids with gating strategy for Clover-positive cells indicated. C) Violin plots showing relative DNA damage repair efficiency across IMR-5 BARD1+/mut cells as quantified with the Clover-LMNA homology-directed repair assay shown in panels A and B. D) Olaparib IC50 values in IMR-5 WT and BARD1+/mut cell lines (left) and in RPE1 WT and BARD1+/mut cell lines (right). E) Cisplatin IC50 values in IMR-5 WT and BARD1+/mut cell lines (left) and in RPE1 WT and BARD1+/mut cell lines (right). F) Relative cytotoxicity of combined olaparib and cisplatin in IMR-5 BARD1+/mut cell lines compared with WT. Each square represents a single dose combination. Blue squares represent drug combinations at which greater cytotoxicity is observed in BARD1+/mut cells; red squares represent drug combinations at which greater cytotoxicity is observed in WT cells. Numbers in corner cells represent the percent of isogenic cells alive compared with WT cells alive at equivalent doses. G) Violin plots of tumor volumes after 2 weeks of olaparib treatment in BARD1+/mut vs WT IMR-5 cell line xenografts. (n = 9-11 mice per cohort). Tumor volumes for olaparib-treated IMR-5 BARD1+/E287fs xenografts measured on day 13. Solid lines denote medians, and dotted lines denote quartiles. H) Proportion of RPE1 WT and BARD1+/Q564* cell nuclei with more than 10 RAD51 foci after treatment with cisplatin. I) Mean RAD51 foci per RPE1 WT and BARD1+/Q564* cell nucleus after treatment with cisplatin. Data in panel C are means (SD) of 3-10 biological replicates of each isogenic cell line, including multiple cell lines with identical BARD1 variants (n = 2 IMR-5 BARD1+/R112*; n = 1 for IMR-5 BARD1+/R150*; and n = 3 for IMR-5 BARD1+/E287fs cell lines). Data in panels D and E are means (SD) of 3-12 biological replicates of each isogenic cell line, including multiple cell lines with identical BARD1 variants (n = 2 IMR-5 BARD1+/R112*; n = 1 for IMR-5 BARD1+/R150*; and n = 3 for IMR-5 BARD1+/E287fs cell lines). Data in panels H and I represent means (SD) of 3 biological replicates for each cell line with a total of 1262 nuclei (range = 245-718 nuclei per replicate) analyzed for RPE1 WT cells and a total of 1596 nuclei (range = 300-905 nuclei per replicate) for RPE1 BARD1+/Q564*cells. *P < .05, **P < .01, ***P < .0001. LMNA = lamin A/C; NS = not statistically significant as measured by t test.