Reconstitution of telomerase activity of TERT mutations in vitro. (A) The presence of the TERT amino acid substitutions reduces reconstituted telomerase activity in vitro. (B) There is no evidence of a dominant-negative effect. Serial dilutions of each transfected lysate were assayed as described in “Quantitative real-time-PCR on primary material.” The control panel contains 2 sets of data: the first triplet contains mock transfection, luciferase plasmid alone, and luciferase with WT TERC plasmid lysates, which were expected to yield no telomerase activity but similar luciferase levels; the second half shows HeLa (+) and heat-inactivated HeLa (−) lysates. T and I denote the start of the TRAP ladder and the corresponding internal control, respectively. (C) Densitometry readings using the triplicate band highlighted with * in panel A were analyzed.³⁴  The means and SD from 3 separate transfection and subsequent TRAP experiments are shown. (D) Luciferase levels (expressed as a percentage of counts/μg protein compared with the WT sample) and (E) TERC levels (normalized using ABL and expressed as a relative percentage compared with the WT sample) were concordant among lysates analyzed from 3 separate transfection and TRAP analysis experiments. The amino acid substitutions are shown above the panels and below the graphs.