Fig 2: Endogenous ATM and KAP1 directly interact in situ exclusively in lytic cells.A. HH514-16 cells were treated with NaB or left untreated and harvested 24 hours later for immunoprecipitation with normal rabbit IgG, rabbit anti-ATM or rabbit anti-KAP1 antibody. Input samples and immunoprecipitated materials were subjected to immunoblotting with indicated antibodies; IgG hc: IgG heavy chain. B. HH514-16 cells were treated with NaB and harvested at 24 hour post-treatment for examination by proximity ligation assay (PLA) with antibodies to ATM and KAP1 (upper 2 panels), anti-ATM antibody and rabbit IgG (middle panel) or anti-KAP1 antibody and goat IgG (lower panel) and subsequent immunostaining with antibody against ZEBRA; arrows: fluorescent foci indicative of intermolecular interactions. Experiments were performed twice.

Fig 3: Chk1 loss triggers replication catastrophe and chromosome mis-segregation by independent pathways.(A) Western blot of ?H2AX, phospho-RPA32 Ser4/8, phospho-KAP1 Ser824, KAP1, Chk1, and CDC45 in U2OS cells, 48 hours after transfection. The left panel shows the chromatin fraction, obtained after an extraction with CSK buffer; the right panel shows whole-cell extracts. H2B and actin were used as loading controls. (B) Percentage (mean ± SD) and representative images of U2OS cells with pan-nuclear ?H2AX staining. More than 2000 cells per sample were analyzed in three independent experiments. Scale bar, 20 µm. As in all graphs in the manuscript, different letters indicate significant differences (see Materials and Methods). (C) Quantification by neutral comet assay of DSB accumulation in U2OS cells (A.U., arbitrary units). The right panel shows representative images of DNA comets. Three hundred cells per sample were analyzed in three independent experiments. The bars on top of the distribution clouds indicate the median. Scale bar, 10 µm. (D) Percentage (mean ± SD) and representative Z-stack images of U2OS anaphase cells with aberrations. About 150 anaphases per sample were analyzed in three independent experiments. The total percentage of aberrant anaphases (bridges plus lagging chromosomes) was used to calculate the statistics. Scale bar, 5 µm. (E) Percentage (mean ± SD) and representative images of binucleated U2OS cells with micronuclei. About 750 binucleated cells per sample were analyzed in three independent experiments. Scale bar, 10 µm. (F) Excess CDC45 in Chk1-deficient cells provokes DSBs and acute replication stress but does not cause chromosome mis-segregation.

Fig 4: Overexpression of RNaseH1 compromises telomere maintenance in ALT cells.(a) Quantifications of fragile telomeres (FTs) and telomere free ends (TFEs) in metaphase spreads from U2OS and HeLa cells infected for 6 days with the indicated retroviruses. Each dot represents the fraction of indicated telomeric aberrations per chromosome end in one metaphase from two to three independent experiments. Chromosome ends (1,200–6,000) were analyzed for each condition. Black bars indicate medians. P-values were computed using the Student’s t-test. *P<0.05, **P<0.001 (indicated sample versus EVmyc sample). (b) Quantifications of leading and lagging TFEs and FTs and of DTSs from CO-FISH experiments performed on U2OS cells infected for 6 days with the indicated retroviruses. Each dot represents the fraction of indicated telomere feature per chromosome end in one metaphase from two to three independent experiments. Chromosome ends (1,900–7,800) were analyzed for each condition. Black bars indicate medians. P-values were computed using the Student’s t-test. *P<0.05, **P<0.001, ***P<0.0001 (indicated sample versus EVmyc sample). (c) Enlarged examples of DNA FISH and CO-FISH experiments on U2OS cells. (d) Partial metaphase spreads from the indicated cells infected with retroviruses for 13 days. (e) Quantifications of FTs and TFEs on metaphase spreads obtained from cells infected for 13 days with the indicated retroviruses. Chromosome ends (1,200–2,400) were analyzed for each condition. Black bars indicate medians. ***P<0.0001 (RH1myv versus EVmyc). (f) Western blot analysis of proteins from cells as in e performed with antibodies against RNaseH1 and total KAP1 (loading control). The asterisk indicates a cross-reacting band. (g) Consequences of altering RNaseH1 (RH1) cellular levels and telomeric hybrids on ALT telomere maintenance. Question mark indicates displaced G-rich telomeric DNA loops, whose existence remains to be verified.

Fig 5: Both the N- and C-termini of ZBRK1 are required for ZBRK1-mediated inhibition of cell proliferation. A, The in vivo interaction between ZBRK1 and KAP1. The lysates of HeLa cells stably expressing EGFP (G), EGFP-ZBRK1 (GZB) or EGFP-fused truncated ZBRK1s (GZBDK and GZBDZ) were immunoprecipitated using an anti-GFP Ab (Roche). Immunoprecipitated proteins were eluted by first boiling in SDS sample buffer and then separating on a 10% SDS-PAGE gel, followed by immunoblot analysis with an anti-KAP1 antibody (Bethyl Laboratories) to detect KAP1 or with an anti-GFP Ab to detect GFP. B, Truncated N- or C-terminus of ZBRK1 loses the ability to inhibit growth. Left, equal numbers of stable HeLa cells expressing EGFP (G), EGFP-ZBRK1 (GZB) or EGFP-fused truncated ZBRK1s (GZBDK and GZBDZ) were seeded to assess growth by crystal violet staining. The viable cell number was determined at the indicated times. Right, the expression of ZBRK1 and its mutants in HeLa cells was analyzed by Western blot. C, Truncated N- or C-terminus of ZBRK1 loses the ability to inhibit proliferation. A focus formation assay was conducted using various stable HeLa cell lines. Columns, focus numbers; bars, mean ± SD. **p < 0.01 compared with control cells (G) using Student’s t-test; NS, no significance compared with control cells (G).