Extended Data Fig. 3. Loss of PTIP rescues fork progression and restart defects in Brca2-deficient B lymphocytes but does not affect RAD51 IRIF.

(a) Genomic instability measured in metaphase spreads from B lymphocytes derived from WT, Brca1^−/−, Rif1^−/−, Brca1^−/−Rif1^−/− mice treated for 6 hr with 10 mM HU (ns, not significant, * P ≤ 0.05, Unpaired t-test). 50 metaphases were analyzed per condition. Experiments were repeated 3 times. (b) Genomic instability measured in metaphase spreads from B lymphocytes derived from WT, Brca2^−/−, Ptip^−/−, Brca2^−/−Ptip^−/− mice treated for 6 hr with 10 mM HU (ns, not significant, * P ≤ 0.05, Unpaired t-test). 50 metaphases were analyzed per condition. Experiments were repeated 3 times. (c) Fork progression in B lymphocytes derived from WT, Brca2^−/−, Ptip^−/−, Brca2^−/−Ptip^−/− mice treated for 1 hr with 0.2 mM HU. Y-axis represents the tract lengths in µm. Numbers in red indicate the mean and standard deviation for each sample (ns, not significant, ****P ≤ 0.0001, Mann-Whitney test). 150 replication forks were analyzed for each genotype. (d) Percentage of restarted replication forks in WT, Brca2^−/−, Ptip^−/−, Brca2^−/−Ptip^−/− B cells treated for 1 hr with 1 mM HU followed by 90 min recovery. 200 replication forks were analyzed for each genotype. (e) Tract lengths of restarted replication forks in WT, Brca2^−/−, Ptip^−/−, Brca2^−/−Ptip^−/− B cells treated for 1 hr with 1 mM HU followed by 90 min recovery. Y-axis represents the tract lengths in µm. Numbers in red indicate the mean and standard deviation for each sample (ns, not significant, ****P ≤ 0.0001, Mann-Whitney test). 150 replication forks were analyzed for each genotype. (f) Western blot analysis for RAD51 levels in WT, Ptip^−/−, Brca2^−/− and Brca2^−/−Ptip^−/− B cell extracts. Tubulin is used as loading control. (g) Quantification of RAD51 foci formation in WT, Brca2^−/− and Brca2^−/−Ptip^−/− B cells upon treatment with 5 and 10 Gy IR and recovery for 2, 4 and 6 hr (n=150 cells analyzed).