Figure 6: 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.