Figure 6. SAMHD1 is critical for telomere integrity in TRF1-depleted but not in aphidicolin-treated cells.

(A–D) Characterization of telomere aberrations on metaphase chromosomes induced by SAMHD1 knockdown in TRF1-proficient and TRF1-deficient HeLa cells. (A) Immunoblotting analysis of knockdown efficiency in cells treated with the indicated shRNAs for 6 d. SAMHD1 and TRF1 are shown on two separate membranes. hnRNPA1 is used as a loading control. Irrelevant lanes have been omitted from the image (dashed lines). (B) Representative metaphase chromosome spreads with telomeres detected by FISH with a Cy3-[CCCTAA]₃ probe (red) and DNA stained with DAPI (gray). White arrows point to typical fragility (smears and multiple telomeric signals), whereas green arrows indicate outside telomeres (i.e., a telomeric signal positioned outside the DAPI signal). (C, D) Quantification of fragile and outside telomeres from cells in (A). 81 metaphases from three independent experiments were analyzed for each condition. The black line represents median. One-way ANOVA with Tukey’s multiple comparisons test (*P < 0.05; **P < 0.01; ****P < 0.0001; ns = not significant). (E, F) Effect of SAMHD1 depletion on telomere structure in aphidicolin-treated HeLa cells. (E) Immunoblot verification of SAMHD1 depletion and DDR induction. Where indicated, the cells were treated with 0.1 μg/ml aphidicolin for 20 h before harvest. (F) Quantification of outside telomeric FISH signal on metaphase chromosome spreads from cells in (E). Shown are data from a representative experiment with ≥17 metaphases and >3,000 telomeres analyzed per condition. Differences are not statistically significant. See also Table S3.