FIG. 3.
Low-O2 microenvironment promotes the nuclear translocation of elevated telomerase reverse transcriptase (TERT) levels in hESCs, despite no significant difference in telomerase activity. hESCs were grown in feeder-free conditions in low (2%) and high (20%) O2 microenvironments to monitor TERT protein abundance and localization, as well as telomerase activity. (A) Representative immunofluorescence images of TERT localization patterns and protein levels in hESCs cultured on Matrigel in 2% and 20% O2. Dashed circles denote single nuclei, and arrows point to areas of extra-nuclear TERT protein abundance within cells. Scale bars=50 μm. (B) Relative telomerase activity in hESCs cultured in 2% or 20% O2 (relative to 20% O2-cultured hESCs). Error bars represent SE, n=3. Letters above histogram bars indicate significant differences (P<0.05). (C) Representative flow cytometric analysis measuring TERT fluorescence levels in H9 hESCs cultured in 20% and 2% O2. TERT fluorescence shown in 2% (blue) and 20% (red) O2 conditions relative to matched isotype controls (black). Viability was determined using 7-AAD staining. (D) Mean fluorescence intensity plots derived from flow cytometry data representing TERT signal of hESCs grown in 2% and 20% O2. Error bars represent SE, n=3, *P<0.05. Color images available online at www.liebertpub.com/scd