Figure 1.

Elongated G-overhang and persistent ExoI-resistant ss DNA at G-rich strand caused by Stn1 depletion in both telomerase-positive and -negative cells. (A) Western blot of Stn1 KD in HeLa and BJ cells, as well as expression of FLAG-tagged RNA-resistant Stn1 (FLAG-MM-Stn1). Cells were infected with retroviral shRNA and selected for puromycin resistance. One day after selection completed, cells were pooled and used for DNA isolation and western blot. Luc: shRNA targeting luciferase. (B) In-gel hybridization of HeLa and BJ cells expressing shStn1. MM-Stn1: RNAi-resistant Stn1 containing mismatch in RNAi targeting region. (C) Quantitation of relative abundance of ExoI-resistant ss G-rich DNA and ExoI-sensitive G-overhangs in HeLa and BJ cells. Signals were normalized to Alu signals. Two-tailed t-test was used to calculate statistical significance. Results were from 5 independent experiments. Error bars: S.E.M. (D) In-gel hybridization with C-rich or G-rich telomeric probe in HeLa cells expressing shRNA. RecJ_f is a 5′→3′ exonuclease that specifically removes 5′ overhangs.