Ric quantification .e.m. (n three).NATURE COMMUNICATIONS | 7:12628 | DOI: 10.1038/ncomms12628 | nature.com/naturecommunicationsARTICLEa100 Clonogenic survival ( )NATURE COMMUNICATIONS | DOI: 10.1038/ncommsbwt Dox: CPT (1 M): ATM-pS1981 10 U2OSGFP- KLHL15 wt (Dox) wt (+ Dox) Y552A (Dox) Y552A (+ Dox) 1 0 1 two 3 4 Camptothecin (nM) five CtIP GFP RPA2-pS4/S8 RPA2 1 2 1hU2OSGFP-KLHL15 Y552A + 1h 1h + 1h95 30 3 4 five six 7cwt Dox RPA signal (a.u.) 39.U2OSGFP-KLHL15 wt + Dox 17.5 Y552A Dox 38.8 Y552A + Dox 42.d1.5 HR (relative to EV) Untreated CPTHEK293 DR-GFP 1.0 ns0.EVBrdU signal (a.u.)31.eight.28.34.0 CtIP TFIIH FLAG 1Y552A130 890.0 FLAG-KLHL15:wtDNA content material (a.u.)Figure four | KLHL15 overexpression results in camptothecin hypersensitivity and defective DNA-end resection. (a) U2OS Flp-In T-REx cells inducibly expressing GFP-KLHL15-wt or GFP-KLHL15-Y552A had been cultivated inside the presence or absence of Dox. Twenty-four hours post induction, cells were treated with the indicated doses of camptothecin and survival was determined just after ten days by colony-formation assay. Data are presented as the imply .d. (n three). (b) Identical cells as inside a had been mock-treated or treated with camptothecin (CPT, 1 mM) for 1 h and lysates have been analysed by immunoblotting working with the indicated antibodies. Asterisks indicate hyperphosphorylated forms of CtIP and RPA2, respectively. (c) Identical cells as in b had been labelled with BrdU (30 mM) for 24 h just before CPT remedy. Cells have been harvested, permeabilized, fixed, immunostained with anti-RPA2 or anti-BrdU antibody and analysed by FACS. Dot plots representing the intensity in the signals for RPA2 or BrdU staining (y axis) against the DNA content material (x axis). Quantification gates were established in untreated samples as well as the percentage of cells inside the gates is indicated. (d) HEK293 DR-GFP cells had been transfected using the I-SceI in combination with the indicated FLAG-KLHL15 expression plasmids and harvested following 48 h for flow cytometry and immunoblot analysis. Data are represented as mean .d. (n three). Statistical analysis had been Cas Inhibitors Reagents carried out applying unpaired, two-tailed t-tests. P values expressed as (Po0.005) have been considered significant. A.u., arbitrary units; FACS, fluorescence-activated cell sorting.DNA-end resection8. Additionally, CtIP contains many quick sequence motifs (Fig. 5a) which might be crucial for CtIP tetramerization368 or for physical interactions with other proteins, such as FANCD2 (ref. 39), PIN1 (ref. 28), BRCA1 (refs 40,41) and CDH1 (ref. 42). Interestingly, by performing MBP-KLHL15 pull-down experiments from HEK293T cell lysates expressing different CtIP constructs, we identified that GFP-CtIP-wt and GFP-CtIP-DN (deleted of amino acids (aa) 15322) interacted with KLHL15, whereas GFP-CtIP-DC1 lacking the entire CTD (aa 79097) did not (Fig. 5b). Furthermore, when precisely the same constructs were cotransfected with FLAG-KLHL15 into HEK293T cells, quantification of protein levels revealed that CtIP-DN showed rather variable abundance, whereas CtIP-DC1 was resistant to KLHL15 overexpression (Supplementary Fig. 5a). Actually, increased protein stability of a C-terminally truncated kind of CtIP has been reported previously43. Consistently, we observed that ubiquitination of CtIP-DC1 in vivo (Fig. 5c) and in vitro (Supplementary Fig. 5b) was decreased compared with CtIP-wt. These benefits indicated that the CTD in CtIP is essential for KLHL15 binding and subsequent ubiquitin-dependent proteolysis of CtIP.To narrow down our search for a putative KLHL15-interaction motif.
