Journal: Communications Biology

Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

doi: 10.1038/s42003-019-0692-z

Figure Lengend Snippet: Metaphase spreads of a , b Hs68 and c , d Hs68 + hTERT cells. DNA was stained with DAPI (blue) and telomeres were stained with PNA-TelC488-conjugated probe (green). a , c Chromosomes were manually counted and labelled in ImageJ. Both Hs68 and Hs68 + hTERT cells counted 46 chromosomes confirming a diploid genome. b , d Telomere staining indicated no telomere-free ends in both Hs68 and Hs68 + hTERT cells. Images were acquired using a Nikon Ti Eclipse Widefield system and deconvolved. Scale bar = 5 µm.

Article Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris.

Techniques: Staining

Journal: Communications Biology

Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

doi: 10.1038/s42003-019-0692-z

Figure Lengend Snippet: 2D analysis of telomeres in interphase Hs68 cells using the ImageJ program. a Example of an interphase Hs68 cell stained with PNA-TelC647-conjugated probe for telomeres (magenta) and DAPI (blue). Grayscale, deconvolved image of PNA TelC647 staining was used for telomere count using the Find Maxima program in ImageJ. b Graph showing average telomere counts per cell using different noise tolerances for Find Maxima ( n = 3 biologically independent experiments, >50 cells per experiment). c Grayscale, raw image of PNA TelC647 staining and grayscale, deconvolved image of PNA TelC647 staining. Intensity-based thresholding (Otsu) of raw image and deconvolved image was performed. Red indicates the foreground signal that is separated from the background when applying the Otsu threshold. d Grayscale, deconvolved image of PNA TelC647 staining in an Hs68 cell. e Inset is an enlargement of the boxed area in panel d . f Plot profile along the purple arrow in the inset in panel e . The intensity graph indicates two spots, but they cannot be separated by thresholding and watershedding. All images were taken using a Nikon Ti Eclipse Widefield system. Scale bars = 5 µm. Source data used for the graphs in b can be found in Supplementary Data .

Article Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris.

Techniques: Staining

Journal: Communications Biology

Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

doi: 10.1038/s42003-019-0692-z

Figure Lengend Snippet: Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris. This was used to determine the location of each telomere within the image. c Visualization of the fluorescence signal of two telomeres (magenta) from above (XY) and from the side (Z). Spots were assigned to the fluorescence intensity that represents telomeric PNA probe. Overlay of the spots with the fluorescence intensity from above (XY) and from the side (Z) shows how accurate the assigned spots reflect telomeric signal. d Overlay of the spots with the fluorescence intensity representing the telomeres in this 3D image of an Hs68 cell. Spots were colour-coded based on volume and visualized from above. Scale bars = 2 µm.

Article Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris.

Techniques: Staining, Fluorescence

Journal: Communications Biology

Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

doi: 10.1038/s42003-019-0692-z

Figure Lengend Snippet: Confirmation of hTERT expression and telomere elongation in the generated cell line Hs68 + hTERT. a Immunoprecipitation of Flag-hTERT in Hs68 and Hs68 + hTERT cells followed by western blot for hTERT protein. Low levels of hTERT were detected in input samples of Hs68, but no hTERT was detected after immunoprecipitation with Flag-antibody. In both input and immunoprecipitated samples of Hs68 + hTERT high levels of hTERT were detected, indicating successful infection with hTERT-plasmid. b RNA expression of hTERT in Hs68 and Hs68 + hTERT measured by qPCR. c Telomerase activity of hTERT in Hs68 and Hs68 + hTERT measured by Telomerase Repeated Amplification Protocol (TRAP). d Relative amount of telomeric DNA in Hs68 and Hs68 + hTERT measured by telo-qPCR. Upon hTERT overexpression, there is a 3.5-fold increase in average telomere length in Hs68 + hTERT compared to Hs68 cells. e Absolute telomere length in Hs68 (~5.3 kb) and Hs68 + hTERT (~18.9 kb) measured by Telomere Restriction Fragments (TRF) analysis. This matched the telo-qPCR data and a 3.5-fold increase in telomere length upon hTERT overexpression. Two-tailed unpaired t -tests were performed, n = 3 biologically independent experiments, **** P < 0.0001. Source data used for the graphs in b and d can be found in Supplementary Data .

Article Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris.

Techniques: Expressing, Generated, Immunoprecipitation, Western Blot, Infection, Plasmid Preparation, RNA Expression, Activity Assay, Amplification, Over Expression, Two Tailed Test

Journal: Communications Biology

Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

doi: 10.1038/s42003-019-0692-z

Figure Lengend Snippet: Hs68 + hTERT cells have longer and bigger telomeres when compared to Hs68. a Frequency distribution of the Intensity Sum of the individual spots assigned in Imaris in 3D. Intensity Sum represents telomere length of the telomeres in Hs68 and Hs68 + hTERT. b Histogram of the logarithm of the Intensity Sum of the spots. c Volume measurements of the individual spots in 3D. Telomeres in Hs68 + hTERT cells are on average twofold bigger when compared to telomeres in Hs68 cells. d , e The coefficient of determination, R 2 , demonstrates a non-linear correlation between intensity and volume of individual spots in Hs68 and Hs68 + hTERT. A weaker R 2 is observed in Hs68 + hTERT cells compared to Hs68. Note the difference in X and Y axes when comparing Hs68 ( d ) with Hs68 + hTERT ( e ). f Telomere count in 2D using the Find Maxima setting in ImageJ and various noise tolerances. g Telomere count in 3D using Imaris ‘Spots’ feature. Hs68 + hTERT cells have on average less telomeres per cell compared to Hs68. Two-tailed unpaired t -tests were performed, n = 3 biologically independent experiments, **** P < 0.0001. Source data used for the graphs can be found in Supplementary Data .

Article Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris.

Techniques: Two Tailed Test