Fig. 5: DNA damage and translocations with ART558.
From: Genetic separation of Brca1 functions reveal mutation-dependent Polθ vulnerabilities
A Brca1+/+, Brca1CC/CC, and Brca1Δ11/Δ11 MEFs were treated with DMSO, ART558, or rucaparib for 24 h, and whole cell extracts were collected to assess γH2ax by western blotting. Representative blots from three independent experiments are shown. B MEFs expressing H2B-mCherry were tracked after 72 h of DMSO or 10 μM ART558 treatments. (Top) The percentage of nuclei with micronuclei present are shown for individual replicates (bar is the median). (Bottom) Individual mitoses were classified as normal or abnormal with mean and S.E.M. percentages for n = 3 biological replicates. Representative images and examples of lagging chromosomes (orange arrow) and anaphase bridges (pink arrow) are shown. Scale bar is equal to 10 μm. See Supplementary Fig. 3b for additional images. C Brca1CC/CC and Brca1Δ11/Δ11 MEFs were treated with thymidine for 16 h and then released into fresh media. DMSO or 10 μM ART558 was added 2 h after release and live cells were imaged for GFP-MDC1 and RPA2-mCherry. MDC1 and RPA2 foci were quantified for 15 individual cells that entered mitosis with foci present over three independent experiments. The mean and S.E.M. number of foci are shown prior to mitosis (P), during mitosis (M), and in daughter cells (D). Representative images are shown. D Translocation frequency in Brca1CC/CC cells was determined after inducing breaks in Rosa26 and H3f3b loci. Cells growing in 96 well plates with DMSO or 10 μM ART558 were compared to wells with no break induction (−). The average percentage of wells with detectable translocation products is shown for n = 4 biological replicates (bar is median). See Supplementary Fig. 3c. E Breakpoint junctions for individual wells from D were sequenced and aligned to the predicted translocation product. (Left) Deletion size was determined for each sequence. (Right) The frequency of deletions (green) and microhomologies (orange) are shown at each coordinate of the predicted translocation product. If multiple sequence traces were detected and could not be deconvolved, those sequences were excluded from analyses, see Supplementary Fig. 3d. Statistical significance was assessed for the indicated comparisons by unpaired, two-tailed t tests. Source data are provided as a Source Data file.
