Fig. 8.

The FAN1/ub-PCNA interaction preserves genome integrity in BRCA2-deficient cells. a Immunoblot of extracts of U2OS cells transfected with siRNAs against FAN1 and/or BRCA2, and treated or mock-treated with aTMS (2 μM; 48 h). The antibodies used are shown on the left. A representative blot of three independent experiments is shown. b PFGE of DNA isolated from cells treated as described in a was performed to visualize DSB induction. Quantification of three independent experiments is shown. Data are represented as mean ± s.d. (n = 3). Statistical analysis was carried out using unpaired, two-tailed t-tests. P values expressed as ** (P < 0.01) were considered significant. c Cell survival of cells transfected as in a was evaluated by colony formation assays upon exposure to aTMS (n = 3). d Colony formation assay of untreated U2OS cells transfected as in a. e Quantification of colony formation observed in d (n = 3). f U2OS cells stably-expressing the indicated FAN1 variant were depleted or not of BRCA2 and their total cell extracts were analysed by immunoblotting with the indicated antibodies. A representative blot of two independent experiments is shown. g Metaphase spreads of cells as in f were analysed for chromatid breaks and radial structures. Representative images of metaphase spreads are shown. h Quantification of chromosomal abnormalities observed in f. Fifty spreads were analysed per single experiment per sample. Statistical significance (P value) was calculated using one-way ANOVA (**** P < 0.0001). i Scheme of the FAN1:ub-PCNA interaction and its biological implications