Loss of PML-IV or NS increases the frequency of telomere damage and abnormalities. Knockdown of PML-IV by siPML4 increases both TIF (53BP1⁺TRF2⁺) and IDF (53BP1⁺TRF2⁻) in U2OS (A) and in HeLa cells (B). (C) The effect of NS and PML-IV knockdown on the telomere integrity was determined by telomere fluorescence in situ hybridization (Tel-FISH), which showed that loss of NS increases the frequency of chromosomes with low telomere signals (Low-TS, arrows) from 27.2% to 42.3% and chromosomal fusion (asterisks) from 3.1% to 5.9%, but has no clear effect on the number of chromosomes with multi-telomeric signals (Multi-TS, M). PML-IV knockdown also increases the frequency of Low-TS chromosomes (44.5%) but does not change the frequency of fusion or Multi-TS event. (D) The telomere length distributions of control (gray), NS-knockdown (green), and PML-IV-knockdown (red) cells were analyzed by Q-FISH. X-axis represents the telomere signal intensity (in arbitrary units). Y-axis indicates the frequency of event. The average telomere intensity (mean) and number of telomeres analyzed (n) were shown. Asterisks in A–C represent P values (see Fig. 1). Bars: (A and B) 5 µm; (C, large panels) 10 µm; (C, small panels) 5 µm.