Fig. 6.

Coilin overexpression is correlated with decreased association of hTR with dyskerin and reduced telomerase activity. (A) Verification of Dox induction of GFP-coilin in the A2F2 and A3B8 lines. Cells were untreated or treated for 48 h with Dox, followed by lysate generation, SDS-PAGE, western blotting and probing with anti-coilin antibodies. (B) Lysate from untreated or Dox-treated (48 h) A2F2 or A3B8 cells was IPed with anti-dyskerin, followed by RNA isolation and qRT-PCR to determine the relative amount of hTR. There is a significant decrease in hTR with dox induction compared to uninduced (P=0.035 for A2F2 line, P=0.008 for A3B8 line, n=4). Error bars represent standard error about the mean. (C) Telomerase activity assays were conducted on equal protein amounts from Dox-induced or uninduced lysate from A2F2 and A3B8 lines, and samples were run on an agarose gel and stained with ethidium bromide (lysis buffer served as negative control). Dox induction of GFP-coilin in A2F2 (lanes 4 and 5) resulted in a 20% decrease in the amount of TRAP signal compared to untreated (n=5, P<0.0005). For the A3B8 line (lanes 2 and 3), Dox induction resulted in a 10% decrease in TRAP signal compared to untreated (n=3, P<0.05). Note that the treatment of non-inducible HeLa cells with Dox did not alter telomerase activity compared to untreated cells (C, right panel).