Figure 4

(A) Analysis of genomic instability in metaphases from B cells treated with PARP inhibitor (1μM). Conditional Brca1 and Lig4 alleles were deleted using pMX-Cre-GFP retroviral transduction to generate B cells that were homozygous for the knockout Brca1ϕ/ϕ and Lig4ϕ/ϕ alleles. Chart (left) shows percentage of metaphases with genomic instability from cells of the indicated genotypes. Chart (right) quantifies the distribution of radial chromosomes, chromatid breaks (ctd) and chromosome breaks (csb) among the aberrant metaphases (n=50 metaphases of each genotype analyzed). (B) Cell survival after treatment with PARP inhibitor. Cultured B cells homozygous for conditional Brca1 and Lig4 alleles were infected with pMX-Cre-GFP retrovirus to produce GFP⁺ knockout Brca1^ϕ/ϕ and Lig4ϕ/ϕ cells. The chart shows the percentage of GFP⁺ cells that were present in B cell cultures of the indicated genotypes five days post-infection. PARP inhibitor treatment (10nM or 1μM) led to disappearance of Brca1^ϕ/ϕ and Brca1ϕ/ϕ Lig4ϕ/ϕ cells from the cultures. (C) Chart showing the percentage of cells with Rad51 foci after ionizing radiation as measured by immunofluorescence (n ≥ 160 cells). (D) Frequency of radial chromosomes in metaphase spreads from Brca1^Δ11/Δ11 p53^+/- B cells cultured for 24 hours with either PARP inhibitor (PARPi) or PARPi + DNA-PKcs inhibitor (DNAPKi).