Fig 2: Enhanced phosphorylation of H2AX and ATM is related to S-phase. (A,B) Analysis of the level of DNA damage in iSML1 iPSCs cells during replication, at either 7 or 14 days. Cells were cultured in doxycycline+ medium as indicated and S-phase cells labelled with EdU for 30 min before sampling. The graphs report the number of ?-H2AX-positive cells (A) or the number of ATM-pSer1981-positive cells (B) in the S-phase population. Data are means, from biological duplicates. Standard errors are not depicted and are <15% of means. Representative images from 14 days are shown below the graphs; right column, ATM activation; left column, H2AX phosphorylation. Scale bar: 10 µm.

Fig 3: Preventing replication-transcription conflicts reduces DNA damage and DDR in SMARCAL1-depleted iPSCs. (A,B) Analysis of accumulation of DNA damage in iSML1 iPSCs after SMARCAL1 downregulation. Four hours before sampling, DRB was added in the indicated samples at 50 µM. The graphs show the percentage of ?-H2AX-positive cells (A) or ATM-pSer1981-positive cells (B). Representative images are shown (bottom). Data are mean±s.e.m from three independent experiments. *P<0.05, **P=0.01, ***P=0.001 (two-way ANOVA test). (C) Experimental scheme for the detection of R-loops by dot blot in genomic DNA (gDNA). (D) Analysis of R-loop accumulation by dot blotting. Genomic DNA was isolated from iPSCs, treated or not with doxycycline (DOX) to induce SMARCAL1 silencing, and then randomly fragmented before being spotted onto a nitrocellulose membrane. The control membrane was probed with anti-RNA-DNA hybrid S9.6 monoclonal antibody. Treatment with RNase H was used as a negative control. (E) Schematic of the labelling strategy to detect replication fork progression (top). Cells were labelled by two consecutive pulses of 30 min with the indicated halogenated nucleotides. DRB was added, where indicated, 4 h before labelling. The graph (bottom) shows a scatter plot of single fork speed from iSML1 iPSCs treated as indicated. (F) The length of the IdU tracts (green; top) was evaluated in at least 150 fibres. Ori, origin of replication. The graph (bottom) shows a scatter plot of the ratio between the right verses left replication tract (green) from bidirectional forks as shown in the scheme on top. Values are from iSML1 iPSCs treated as indicated. Data are mean±s.e.m. ****P<0.0001 (Mann–Whitney test). ns, not significant. Scale bars: 10 µm.

Fig 4: Kaplan-Meier analysis of overall survival (OS) with the combination of two prognosis factors. (A,B) OS curves for the combination of ATM and BRCA1 in (A) the KMUH cohort (n for both high: 26, both low: 31, others: 43) and in (B) TCGA cohort (atypical and classic molecular subtypes, n for both high: 41, both low: 23). (C,D) OS curves for the combination of ATM and (C) tumor size (n for ATM > 0.3 and T1–2: 41, ATM < 0.3 and T3–4: 20, others: 39) or (D) lymph node involvement (n for ATM > 0.3 and N0: 43, ATM < 0.3 and N1–3: 17, others: 40) in the KMUH cohort. (E,F) OS curves for the combination of BRCA1 and (E) tumor size (n for BRCA1 > 1 and T1–2: 19, BRCA1 < 1 and T3–4: 29, others: 52) or (F) lymph node involvement (n for BRCA1 > 1 and N0: 18, BRCA1 < 1 and N1–3: 23, others: 59) in the KMUH cohort.

Fig 5: Differential phosphorylation responses of DNA damage response-related proteins. a Heatmap of the level of differential phosphorylation at various sites within the DDR-related proteins: Atm, Chek2, Atr, Chek1, Topbp1, Brca1, and Brca2. b Heatmap of the level of phosphorylation for 50 phosphosites with an (S/T)Q motif in proteins selected using “DDR” in the “Keywords” annotation from UniProt. c Fraction of apoptotic/necrotic cells as determined by Annexin V staining at 24 h after initiation of stressor exposure in the presence or absence of indicated kinase inhibitors. Statistics: one-way ANOVA test between cells treated with indicated stressors with or without the indicated inhibitors (****p-value < 0.001; ***p-value = 0.0006; n.s. not significant)