NS depletion triggers telomeric and nontelomeric DNA damage in ALT and TA⁺ cells, and decreases the formation of APB in ALT cells. (A) Damage on the telomere (TIF) and interstitial chromosome (IDF) was detected by the 53BP1⁺TRF2⁺ and 53BP1⁺TRF2⁻ foci, respectively, on 3D-reconstructed confocal images. The bottom and right images represent the stacked images along the X-Z and Y-Z axis, respectively. NS knockdown (NS-KD) by siNS increases both TIF and IDF in U2OS (ALT) cells. (B) NS-KD also increases TIF and IDF in HeLa (TA⁺) cells. (C) Transfection of a TRF2-dominant mutant (TRF2-DN, also known as TRF2^ΔBΔM) significantly increases the number of TIF and the percentage of TIF associated with APB (PML⁺53BP1⁺TRF1-GFP⁺ over 53BP1⁺TRF1-GFP⁺ foci). The TRF2-DN–induced increase of APB-occupied TIF is reduced by NS-KD (yellow bars). In contrast, the percentage of undamaged telomeres occupied by APB is hardly affected by TRF2-DN transfection or NS-KD (gray bars). (D) NS-KD reduces the formation of APB (TRF2⁺PML⁺) in U2OS cells. Y-axis indicates the percentage of APB among PML bodies (APB/PML). (E) A nucleoplasmic mutant of NS, NSdB, is distributed predominantly in the nucleoplasm (E1) but still retains the ability to bind TRF1 in coIP experiments (E2). Overexpression of wild-type NS or NSdB can both promote the APB formation in U2OS cells (E3). Fib, fibrillarin. *, P < 0.01; **, P < 0.001; ***, P < 0.0001. Bars, 5 µm.