Fig 1.
RNAscope detects TERC and TERT in heterogeneous cell populations.
A. Representative images showing HeLa cells and HeLa cells overexpressing TERT (HeLa TERT), stained for TERT mRNA and TERC. Each of these cell types was infected with a lentiviral plasmid expressing the TSQ1 mutant TERC or a vector control (vec). Scale bars denote 10μM. B. qPCR to measure TERT and TERC expression in HeLa vec, HeLa TSQ1, HeLa TERT vec and HeLa TERT TSQ1 cells. In each case, levels are expressed as fold change over control HeLa vec levels. C. Frequency distribution of RNAscope TERT foci in populations of HeLa and HeLa TERT cells. The data is from 3 sets of biological replicates containing 100 cells each for either cell type.
Fig 2.
A. RNAscope for TERT expression in telomerase negative (MRC5 and WI38 fibroblast) and positive (HCT116 and LOX) cell lines. B. Frequency distribution of TERT RNAscope signals within populations of MRC5, WI38, HCT 116 and LOX cells. The data is from 3 sets of biological replicates each containing 100 cells for all cell types. Scale bars denote 10μM.
Fig 3.
Combined RNAscope/PNA-FISH can be used to detect TERT expression and telomere elongation in single cells.
Cells were assayed 7 days post-TSQ1 or vector infection. They were imaged via fluorescence for wild-type telomere (green) and TSQ1 (red) signal and then bright-field for the brown TERT DAB reaction product. TSQ1 spots co-localizing at telomeres are marked with arrows. Representative images are shown and scale bars denote 10μM.
Fig 4.
Quantification of TERT expression and telomere elongation in single cells.
Swarm plots showing the number of TSQ1 spots co-localizing with telomeres in HeLa cells and HeLa cells overexpressing TERT. 171 cells from at least 2 separate experiments were assayed for each group. Means (horizontal black lines) and SEM (red lines) are shown for each population. P values (Mann-Whitney test) are provided and significant differences are marked with asterisks.
Fig 5.
Correlation of TERT expression levels with incorporation of TSQ1 repeats in HeLa and HeLa POT1-ΔOB cells.
A. Representative images of HeLa cells with robust TERT expression and either robust TSQ1 incorporation (upper panel of images) or no TSQ1 incorporation (lower panel of images). Arrows indicate TSQ1 spots that co-localize with telomeres. Enlarged images of the boxed regions are in the lower right-hand corner of each corresponding image. Scale bars denote 10μM. B Scatter plot demonstrating the correlation between TSQ1 incorporation and TERT expression in HeLa (left panel) and HeLa POT1-ΔOB cells (right panel). 171 cells from at least 2 separate experiments were assayed for each group. C. Swarm plots showing number of TSQ1 spots co-localizing with telomeres (left panel) and RNAscope TERT expression (right panel) in HeLa cells and HeLa cells overexpressing POT1-ΔOB. 171 cells from at least 2 separate experiments were assayed for each group. Means (horizontal black lines) and SEM (red lines) are shown for each population. P values are provided and significant differences are marked with asterisks, with ns denoting a non-significant difference. Of note, the HeLa cells quantified in this figure are the same as the cohort shown in Fig 4.