Figure 7(A) MiDAS analysis was performed in U2OS WT and PIF1-KO cells. Cells were treated with APH (0.4 µM) and RO-3306 (7 µM), and then released into fresh medium containing EdU (20 µM) and Colcemid (0.1 µg/ml) as described in Materials and Methods. The image of EdU incorporation on metaphase chromosomes is shown (left) and EdU foci formed on each metaphase spread were quantified (right). ~100 metaphase spreads were analyzed in each sample. Scale bars, 10 μm. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (B) U2OS (HR-Flex1-STGC-1541) cells expressing FLAG-PIF1 or vector were treated with or without APH (0.4 μM, 22 hr) in the presence of RO3306 (7 μM), and anti-FLAG ChIP was performed 30 min after release from drug treatment and analyzed by qPCR. Enrichment of FLAG-PIF1 at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in cells expressing vector as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (D) Metaphase spread of HCT116 WT and FANCM-KO cells was performed with or without expressing PIF1 shRNA or shRNA vector (Ctrl) before and after APH treatment (0.4 μM, 24 hr). Representative images of metaphase spread are shown (left), and overall chromosome breaks and gaps per cell are quantified (right). Scale bars, 5 μm. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (F) U2OS (EGFP-Flex1-BIR-5086) cells expressing PIF1 WT or E307Q, L319P mutants with endogenous PIF1 silenced by shRNA were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells by HU treatment was quantified by FACS analysis 3 days after HU removal. . Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (G) U2OS PIF1-KO cells expressing PIF1 WT or E307Q, L319P mutants were treated with the indicated concentrations of HU for 72 hr, and cell viability assays were performed. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (H) U2OS PIF1-KO cells expressing PIF1 WT or E307Q, L319P mutants were labeled with CldU for 30 minutes, followed by 2 mM HU treatment for 2 hr and IdU labelling for 30 minutes, and processed for DNA fiber analysis. The percentage of replication restarted DNA fibers is calculated. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. (I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.