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. 2019 Mar 29;15(3):e1008049. doi: 10.1371/journal.pgen.1008049

Fig 4. Sensitivity of BARD1 variants to ionizing radiation.

Fig 4

(A) Endogenous-only cells and cells expressing BARD1 WT, A460T, P707S, G753D, and V767fs were treated with control, BARD1 3'UTR or BRCA1 3’UTR siRNA and X-ray irradiated at doses of 1, 2, 4 and 6 Gy. Colonies were counted after 12 days of growth followed by staining with crystal violet. Results in each experiment (±S.E.M.) were carried out in triplicate and converted to logarithmic scale. In each plot, the dashed line indicates control siRNA (siCON), and the solid line indicates depletion with the BARD1 (siBARD) or BRCA1 (siBRCA) 3'UTR-targeted siRNA. The Student’s t-test was done to examine the growth of variant and endogenous-only cell lines treated with BARD1 or BRCA1 siRNA relative to variant and endogenous-only cells treated with control siRNA (indicated by asterisks; * = p < 0.05, ** = p < 0.01). Cells expressing the four BARD1 variants, as well as endogenous-only cells depleted of BARD1 and BRCA1, were significantly different from variant and endogenous-only cells treated with control siRNA across most irradiation doses. (B) Results from panel A are shown for the BARD1 WT and variant cell lines treated with BARD1 3'UTR siRNA and endogenous-only cells treated with BARD1 or BRCA1 3’UTR siRNA. A Student’s t-test was done to compare the colony formation of BARD1 variant and BRCA1 or BARD1-depleted cell lines to BARD1 WT cells (indicated by asterisks). Colony counts of variants and BRCA1 or BARD1-depleted cell lines were significantly different (p < 0.01) from WT at all concentrations except siBARD 6 Gy (p < 0.05) and P707S 1 Gy, G753D 1 Gy, and G753D 2 Gy (p > 0.05). (C) An examination of the expression of endogenous BARD1 and BRCA1 in treated BARD1 variant-expressing and endogenous-only cell lines. Cells treated with BARD1 3'UTR siRNA had depleted endogenous BARD1 expression, while HBT BARD1 was unaffected. Cells treated with BRCA1 3’UTR siRNA showed depletion of endogenous BRCA1 and BARD1 expression. In all cases, the band representing the expression of the variant HBT BARD1 was denser than the faster migrating band from the endogenous BARD1 protein.