Figure 4: KLHL15 overexpression leads to camptothecin hypersensitivity and defective DNA-end resection.

(a) U2OS Flp-In T-REx cells inducibly expressing GFP-KLHL15-wt or GFP-KLHL15-Y552A were cultivated in the presence or absence of Dox. Twenty-four hours post induction, cells were treated with the indicated doses of camptothecin and survival was determined after 10 days by colony-formation assay. Data are presented as the mean±s.d. (n=3). (b) Same cells as in a were mock-treated or treated with camptothecin (CPT, 1 μM) for 1 h and lysates were analysed by immunoblotting using the indicated antibodies. Asterisks indicate hyperphosphorylated forms of CtIP and RPA2, respectively. (c) Same cells as in b were labelled with BrdU (30 μM) for 24 h before CPT treatment. Cells were harvested, permeabilized, fixed, immunostained with anti-RPA2 or anti-BrdU antibody and analysed by FACS. Dot plots representing the intensity of the signals for RPA2 or BrdU staining (y axis) against the DNA content (x axis). Quantification gates were established in untreated samples and the percentage of cells within the gates is indicated. (d) HEK293 DR-GFP cells were transfected with the I-SceI in combination with the indicated FLAG-KLHL15 expression plasmids and harvested after 48 h for flow cytometry and immunoblot analysis. Data are represented as mean±s.d. (n=3). Statistical analysis were carried out using unpaired, two-tailed t-tests. P values expressed as ** (P<0.005) were considered significant. A.u., arbitrary units; FACS, fluorescence-activated cell sorting.