Protocol to stimulate and delineate alternative lengthening of telomeres in human U2OS cells

This protocol describes how to stimulate the formation of ALT-associated PML bodies (APBs) and ALT activity by tethering PML-IV to telomeres in human U2OS cells. The method involves using an ABA-inducible system to tether PML-IV to telomeres, promoting APB formation, and then visualizing proteins and DNA synthesis in APBs using techniques like immunofluorescence and fluorescence in situ hybridization. This allows researchers to analyze telomere clustering dynamics, observe the recruitment of DNA repair proteins to APBs, and measure telomere DNA synthesis during alternative lengthening of telomeres (ALT).

• 1
  ABA-dependent tethering of PML-IV to telomeres induces telomere clustering.
• 2
  BLM, a protein critical for ALT, is colocalized with telomeres after 14 h ABA treatment.
• 3
  Telomere DNA synthesis in non-S phase cells is enhanced by ABA.