Fig. 6. Cells expressing BRCA2 variants S206C and T207A display reduced stability of kinetochore–microtubule attachments and misaligned chromosomes.

a Top: Scheme of the double thymidine block procedure used to synchronize the DLD1 cells for the analysis of chromosome alignment. Bottom: Quantification of misaligned chromosomes outside the metaphase plate in DLD1 BRCA2 deficient cells (BRCA2^−/−) and BRCA2^−/− cells stably expressing BRCA2 WT or the S206C and T207A variants. n indicates the total number of cells counted for each clone from two (BRCA2^−/−, S206C, and T207A) and four (BRCA2 WT) independent experiments. Statistical significance of the difference was calculated with unpaired two-way ANOVA test with Tukey’s multiple comparisons test, the p-values show the significant differences. b Representative images of the type of chromosome alignment observed in cells quantified in (a), scale bar represents 10 µm. c Top: Scheme of the synchronization procedure for the analysis of cold stable microtubules in the BRCA2-WT and T207A stable clones. Bottom: Representative images of cold stable microtubules in cells expressing BRCA2 WT or the T207A variant. Cells treated according to the scheme were co-stained with α-tubulin and CREST, as centromere marker and DNA was counterstained with DAPI. Scale bar represents 5 µm. A zoom-in inset in the images show representative kinetochore–microtubule attachments in each cell line. d Quantification of the relative intensity of α-tubulin normalized to the one in BRCA2 WT cells in (c). The data represent the results from two independent experiments from a total of 122 (WT) and 123 (T207A) cells. The red line in the plot indicates the median intensity (95% CI), each dot representing the intensity for one cell. For statistical comparison of the differences between the samples we applied Mann–Whitney two-tailed analysis, the p-values show significant differences. Source data are available as a Source Data file.