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	Fig. 1. Mouse LigI-deficient cells show endogenous LigIII and XRCC1 accumulation at PCNA- and BrdU-stained foci. a Endogenous LigI (red) was co-immunodetected with BrdU incorporation sites (green) in proliferating wild-type (LIGI+/+) and LIGI knockout (LIGI−/−) mouse cells. b Co-immunodetection of endogenous LigIII (red) and BrdU incorporation site or PCNA staining (green) in late-S phase LIGI+/+ and LIGI−/− cells. LigIII signal overlapping with BrdU or PCNA signal is observed in LIGI−/− cells with typical late-S phase BrdU or PCNA staining (enlarged images below showing examples of late-replication structures ring-shape or horseshoe staining). The thresholded images were obtained as described in “Materials and methods”. Scale bars 5 μm. c Percentages of nuclei with more than two late-replication structures in the depleted cells. For each double staining combination, more than 40 nuclei were analyzed per cell line. Representative images from cells co-immunostained for endogenous XRCC1 and BrdU or XRCC1 and PCNA are presented in ESM Fig. 1b, c
	Fig. 2. LigIII and XRCC1 proteins accumulate at PCNA and BrdU-stained foci in the 46Br.1G1 cells derived from a LigI-deficient patient or in human cells depleted in LigI by shRNA targeting. a Detection of endogenous LigIII, XRCC1 or LigI (in red) and BrdU incorporation sites (green) in AS3WT2 human cells derived from a healthy donor and 46Br.1G1 cells obtained from a LigI-deficient patient. Co-localization of the LigIII, XRCC1 or LigI proteins and the BrdU-positive replication foci appears as yellow spots in the merged images. b Endogenous LigIII (red) and PCNA foci (green) in control HeLa cells (HeLaCTL), LigI-depleted HeLa cells (HeLa LigIKD) or LigIII-depleted HeLa cells (HeLa LigIIIKD). The co-localization of LigIII protein and PCNA foci appears as yellow spots in the merged images. Scale bar on all merged images, 5 μm. c Thresholded merge images were used to determine the percentage of nuclei from AS3WT2 and 46Br.1G1 cells lines (left panel) and from HeLaCTL and HeLa LigIKD cell lines (right panel) with more than two typical mid- or late-replication structures are presented for the indicated co-staining
	Fig. 3. Increase in chromatin-bound LigIII and XRCC1 in LigI-depleted cells. a Asynchronously growing HeLa cell populations expressing control, LIGI, or LIGIII shRNAs from the pEBV episomal plasmid were harvested and divided into two fractions: whole cell extracts (WCE) and extracts prepared with the indicated Triton concentrations to yield the insoluble fraction. The WCE preparation (left panel) and Triton-resistant extracts (right panel) were then analyzed by Western blotting with the indicated antibodies. *Non-specific band. b WCE (left panel) and the 0.5 % Triton-resistant extracts (right panel) were also prepared from lentiviral-transduced HeLa, MCF7, and U2OS cell populations expressing the indicated shRNA and then analyzed by Western blotting with the indicated antibodies. c WCE (left panel) and 0.5 % Triton-resistant (right panel) LIGI+/+ and LIGI−/− mouse cell line and AS3WT2 and 46Br.1G1 human cell line extracts analyzed by Western blotting with the indicated antibodies
	Fig. 4. LigIII is required for the viability and efficient DNA replication of the LigI-deficient cells. a Effects of LigI-depletion on the plating efficiency in HeLa, MCF7, and U2OS cells following lentiviral transduction with a LIGI shRNA (shLigI) were compared to the respective control (shCTL) cell populations. b The control shRNA (shCTL) or LIGI shRNA (shLigI) molecules were introduced by lentiviral transduction of HeLa cells expressing either a control (CTL), LIGIII (LigIIIKD), XRCC1 (XRCC1KD), or PARP1 (PARP1KD) shRNA from the pEBV episomal plasmid. The plating efficiency of the cell populations expressing the indicated pair of shRNAs was compared to that of the HeLaCTL-shCTL cells expressing the control shRNAs. c The LigI, LigIII, XRCC1, and PARP1 protein levels were analyzed by Western blotting. d Two days post-infection, LigI-depletion was verified at the cellular level by immunofluorescence (red) and the replicating cells were detected by BrdU incorporation (green). e Percentages of BrdU-positive cells detected in the indicated HeLa cells populations following a 30-min BrdU pulse. The values ± SD represent the mean of two experiments for which at least 150 nuclei per experiment were scored
	Fig. 5. LigI and LigIII cooperate in the suppression of SCEs. A representative image of metaphase spreads prepared for SCE analysis from HeLa cells depleted in both LigI and LigIII. The histogram on the left shows the average SCE frequency per chromosome in the different cell types calculated from 37 to 50 metaphases for each cell lines ***p < 0.0001
	Fig. 6. Telomere instability in LigI-depleted HeLa cells. a Percentages (mean ± SD) of sister telomere fusions per chromosome determined from Telo-FISH-stained metaphase spreads of the indicated transduced cells and an example of a chromosome with a sister telomere fusion (white arrow). b Percentages of chromosomes with sister telomere fusion determined from Telo-FISH-stained metaphase spreads for the indicated cell populations. c Example of sister telomere fusion (yellow, white arrow) involving both leading (green) and lagging (red) strand telomeres. d Percentage of chromosomes with lagging strand telomere capture in the different cells indicated (left) and example of lagging strand telomere capture detected by CO-FISH (white arrow). e Percentage of chromosomes with leading strand telomere capture in the different cells indicated (left) and example of leading strand telomere capture detected by CO-FISH (white arrow). Percentages were calculated from 32–42 metaphases for each cell line. Statistical significance levels are noted as follows: ***p < 0.0005, **p < 0.005, and *p < 0.05
	Fig. 7. LigI-defect promotes an increased number of mitotic abnormalities. a Representative time-lapse images of HeLa H2B-GFP LigIKD mitosis. The videos of these mitotic events were synchronized from the onset of prophase. The numbers indicate the time in minutes, the arrows point to bridged DNA (middle row) and misaligned chromosomes (lower row). b Percentage of mitoses in control (open bar) and LigIKD HeLa H2B-GFP (closed bar). c Percentage of abnormal mitoses in control and LigIKD HeLa H2B-GFP. d Percentage of abnormal mitoses with anaphase bridges. e Percentage of abnormal mitoses with misaligned chromosomes. A total of 257 and 270 mitoses were analyzed, respectively. Statistical significance levels are noted as follows: NS not significant, *p < 0.02 and (***p < 0.0001)

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