**Journal:** Cell reports

**Article Title:** BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

**doi:** 10.1016/j.celrep.2024.114006

**Figure Lengend Snippet:** Related to Fig. S3. A) A schematic for rescuing HDR in Brca1 mutant cells by ablation of 53bp1. Relevant substrates that can be repaired by HDR or NHEJ are shown. B) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype to dual allele targeting in the control. Statistical analysis using one-way ANOVA, except for the comparison between wt ctrl and 53bp1−/−, evaluated with a two-tailed, unpaired student’s t-test.; wt ctrl n = 9, Brca1tr/+ n = 3, 53bp1−/− n = 5, Brca1tr/tr n = 3, Brca1tr/tr53bp1+/− n = 2, Brca1tr/tr53bp1−/− n = 4. C) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype and data was analyzed by one-way ANOVA. D) Immunofluorescence and quantification for phospho-H2AX (S139). Foci counted on reprogramming day 5 (≥410 cells/genotype), statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho RPA(S33) on reprogramming day 5. ≥140 cells per genotype, analyzed by one-way ANOVA. For control and BRCA1, images are identical for Fig. 3 and Fig. 4 for panels c and d. F) Cell proliferation analysis with CFSE dye on reprogramming day 5. Statistics with one-way ANOVA.; wt ctrl n = 4,, 53bp1’/- n = 5, 53bp1−/− n = 3, Brca1tr/tr n = 2, Brca1tr/trSmarcal1−/− n = 3, Brca1tr/tr 53bp1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5, analyzed by one-way ANOVA.; wt ctrl n = 3, 53bp1+/− n = 3, 53bp1−/− n = 2, Brca1tr/tr n = 3, Brca1tr/tr 53bp1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. Number of AP positive colonies is shown as a ratio to wild type, analyzed with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, 53bp1+/− n = 7, 53bp1−/− n = 8, Brca1tr/tr n = 12, Brca1tr/tr 53bp1−/− n = 7. I) Quantification of Nanog positive colonies in the indicated genotypes, analyzed with one-way ANOVA.; n = 3 for each genotype. J) AP staining and reprogramming efficiency quantification in human 1023 fibroblasts from adult skin biopsy in control and 53BP1 knockdown (KD) conditions. Cells were fixed on day 25 post reprogramming factor transduction, statistical analysis using an unpaired, two-tailed student’s t-test.; ctrl n = 16, 53BP1 KD n = 16. Samples with n=2 are not used for statistical comparisons. All numbers indicated are biological replicates.

**Article Snippet:** Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

**Techniques:** Mutagenesis, CRISPR, Control, Comparison, Two Tailed Test, Immunofluorescence, Marker, Staining, Knockdown, Transduction