Fig. 6. FANCL promotes CtIP recruitment to DNA double-strand breaks.
a, b U2OS FANCLKO and wild-type control cells (Con.) were exposed to 10 Gy ionizing radiation (IR), followed by IF microscopy to detect CtIP foci in S-phase (EdU + ) nuclei. Panel (a) shows representative images and panel (b) shows the quantification (scale bar = 10 μM). Plotted are the data from all biological repeats. Each gray or green dot represents an individual nucleus, the total number of nuclei analyzed are indicated in italics, and black dots are the median for each biological repeat (n = 3 independent biological replicates; mean ± SEM; one-way ANOVA with post-hoc Dunnett’s). EV empty vector, WT wild-type, LD ligase-dead. c, d IF-microscopy of UV-A laser micro-irradiated cells. Panel (c) shows representative images, panel (d) shows the quantification (scale bar = 10 μM). Plotted are the data from all biological repeats. Each gray or green dot represents an individual track, the total number of tracks analyzed are indicated in italics, and black dots are the median for each biological repeat (n = 3 independent biological replicates; mean ± SEM; ratio paired t test, two-sided). PKi = DNA-PKcs inhibitor NU7441, 2 μM. e Western blot analysis of CtIP overexpression in the cells described in panel (f). f Indicated HEK 293T + DSB-Spectrum_V3 cell lines were transfected to express either CtIP or an empty vector control, together with Cas9 and the sgRNA targeting the BFP gene in the reporter locus. Next, cells were analyzed by flow cytometry to determine the frequency of repair by each of the three indicated pathways. Data were normalized to the Con.+ EV (n = 4 independent biological replicates; mean ± SEM; One-way ANOVA with post-hoc Dunnett’s). HR homologous recombination, mut-EJ mutagenic end-joining, SSA single-strand annealing. g Model depicting how FANCL/Ube2T promotes the repair of DSBs by homologous recombination. See main text for details. Source data are provided as a Source Data file.