Figure 5: RNaseH1 depletion affects leading-strand telomeres in U2OS cells.

(a) Western blot analysis of total proteins from U2OS and HeLa cells infected with retroviruses expressing myc-tagged RNaseH1 (RH1) or catalytically dead RNaseH1 (RH1^CD) or with empty vector control retroviruses (EV) and transfected with the indicated siRNAs. Experiments were performed 6 days after infections and 3 days after siRNA transfections. Endogenous and myc-tagged RNaseH1 proteins were simultaneously detected using anti-RNaseH1 antibodies. The asterisk indicates a cross-reacting band. Total KAP1 was detected to control for loading. (b) Quantifications of TFEs in the indicated cells. Each dot represents the fraction of TFEs per chromosome end in one metaphase from two to three independent experiments. Chromosome ends (2,700–3,640) were analyzed for each condition. Black bars indicate medians. P-values were computed using the Student’s t-test. ***P<0.0001 (indicated sample versus siCtrl-transfected EVmyc sample). (c) Examples of FISH experiments performed on metaphase spreads from the indicated cells. Telomeric DNA is in red and DAPI-stained total DNA in blue. Arrows point to TFEs. (d) Quantifications of leading and lagging TFEs from CO-FISH experiments performed on the indicated U2OS cells. Each dot represents the fraction of TFEs per chromosome end in one metaphase from two to three independent experiments. Chromosome ends (1,900–3,900) 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 siCtrl-transfected EVmyc sample). (e) Examples of CO-FISH experiments performed on U2OS cells. Lagging-strand telomeres are in red, leading-strand telomeres are in green and DNA is in blue. Example of normal telomeres and TFEs are shown.