NS promotes the colocalization of PML bodies and SUMOylated TRF1 foci and the binding between PML-IV and SUMOylated TRF1 proteins. (A) A SUMO-conjugated TRF1 (TRF1-So) was created by fusing SUMO1 to the C terminus of TRF1 (left). The SUMO1 fusion was deleted of its six C-terminal residues, including the di-Gly motif indicated by the X-mark. SUMO-compromised TRF1 constructs were created by mutating six Lys residues to Arg on TRF1 (TRF1R6) or TRF1-So (TRF1R6-So). Respectively, TRF1-So and TRF1R6 mutants mimic the SUMOylated and deSUMOylated state of TRF1 in their abilities to form APB in U2OS cells (right). (B) The percentage of APB among PML bodies (APB/PML) of TRF1-So is decreased by NS-KD (shNS) and increased by NS overexpression (NS-OE), but not affected by GNL3L perturbation. (C) CoIP of FLAG-tagged TRF1 (or TRF1-So) and Myc-tagged PML (III, IV, or VI) reveals a specific interaction between PML-IV and TRF1-So. (D) Real-time RT-PCR confirms the knockdown efficiency of PML-IV–specific siRNA (siPML4) on the PML-IV transcript but not on the PML-I transcript in U2OS cells. (E) Knockdown of PML-IV decreases the APB/PML percentage of U2OS cells from 60.3% to 46.1% without changing the total number of PML bodies. (F) Knockdown and overexpression experiments showed that NS plays a role in promoting the binding between SUMOylated TRF1 and PML-IV. (G) PML-IV knockdown completely abolishes the NS-dependent increase of APB formation of SUMOylated TRF1. (H) TRF2-DN–triggered telomere damage induces APB formation in HeLa (TA⁺) cells. This increase is significantly attenuated by depletion of NS or PML-IV.