Extended Data Fig. 4. Depletion of PTIP rescues the lethality of Brca2-deficient ES cells.

(a) Western blot analysis for PTIP levels in WT and two different clones of Brca2^−/− ES cells electroporated with shPtip. (b) Southern blot analysis for determination of Brca2 deletion in surviving clones electroporated with shPtip. Probes distinguishing the Brca2-flox/⁻ allele (4.8 kb) (upper band) and Brca2 KO allele (2.2 kb) (lower band) were used. * indicates surviving ES cell clones with Brca2 deletion and simultaneous down-regulation of PTIP (12/96 Brca2-deleted colonies were found with shPtip1 and 3/60 colonies were found with shPtip2). Genotyping was confirmed by PCR (Fig. 2b). (c) Upper panel shows representative FACS profiles of WT and Brca2^−/−/shPtip ES cells electroporated with either pDR-GFP plasmid only (control) or pDRGFP and I-SceI expressing vector for 48 hr. Gene conversion of the pDR-GFP construct by HR is determined by the percentage of GFP-positive cells (FL1, green-detection filter; FL2, red-detection filter). Lower graph shows the quantification of the percentage of GFP-positive cells in WT and Brca2^−/−/shPtip ES cells across 3 independent experiments. ns, not significant, *P ≤ 0. 05, Unpaired t-test. (d) Sister chromatid exchange (SCE) analysis in WT and Brca2^Y3308X hypomorphic ES cells. Twenty metaphases were analyzed per condition; experiments were repeated 3 times. (e) WT and Brca2^Y3308X hypomorphic ES cells were preincubated with mirin, EdU-labeled for 15 min and treated with 4 mM HU for 2 hr. Proteins associated with replication forks were isolated by iPOND and detected by Western blotting with the indicated antibodies. (f) SCEs in WT, Brca2^−/− and Brca2^−/− Ptip^−/− B cells either untreated or treated overnight with 1 µM PARPi or with 0.5 µM cisplatin (ns, not significant, *P ≤ 0.05, ** P ≤ 0.001, Unpaired t-test). Twenty metaphases were analyzed per condition; experiments were repeated 3 times.