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. 2018 Apr 1;29(7):869–880. doi: 10.1091/mbc.E17-11-0637

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© 2018 Schmidt et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 2: — Halo-telomerase elongates telomeres in vivo. (A) Experimental design. Overexpression of TERT (star represents tag) will increase telomerase levels in HeLa cells by driving the assembly of free TR into telomerase RNPs. Owing to its higher levels, exogenous tagged TERT will outcompete endogenous TERT for assembly with TR. (B) Western blots of cell lysates (Input) and TERT immunopurified (TERT IP) from cell lines overexpressing various TERT alleles probed with an anti-TERT antibody. Cell lysates were probed with an anti-beta-Actin antibody as loading control. (C) Telomere length analysis of polyclonal HeLa cell lines stably overexpressing various TERT proteins by Southern blot of telomeric restriction fragments. Each rate of telomere extension was calculated relative to the previous time point recorded. (D) Telomere length analysis of single-cell clones of HeLa cells stably overexpressing various TERT proteins by Southern blot of telomeric restriction fragments. In C and D, the dashed line represents the mean length of parental cell telomeres. (E) Quantification of the rate of telomere length change by averaging the telomere length of all single-cell clones (n = 5–8, see panel D), calculating their length relative to those of the parental HeLa cells (see panel C) and dividing by the number of population doublings between introduction of the TERT transgene and analysis of telomere length.