Fig. 5. Telomere aggregates are associated with -H2AX foci but not with TERT. (A) Telomere aggregates do not overlap with TERT in senescent cells. The 81y cells were transduced at passage 6 with Trf2-citrine lentiviral vector, and immunodetection with anti-TERT and anti-lamin-A antibodies was carried out on cells at passage 7. Left panels show maximum projections of TERT (red) and lamin A (blue) in normal (i) or senescent (ii,iii) cells. Right panels show maximum projections of TRF2 and TERT in the x-y axis. Fluorescence intensity graphs on the y-z or x-z axis show overlap in distribution (indicated with white arrows). Gray arrows indicate large TRF2 aggregates. (B) TERT colocalizes with intranuclear structures of the lamina in senescent hMSCs. Immunofluorescence with anti-TERT (green) and anti-lamin A (red) antibodies was carried out on hMSCs at passage 9. (i) Wide-field fluorescence microscope images show a correlation between lamina deformed shape and reduced expression level of TERT in senescent cells. Scale bars are equal 15 µm. (ii,iii) Cross-sections (x-y axis) of confocal images show a cell with a normal ellipse lamina structure (ii) or a distorted lamina shape (iii). The overlap in spatial localization between lamin A (red) and TERT (green) is indicated in the intensity plots in the merged images. (C) Large telomere aggregates overlap with -H2AX in cells with a folded lamina structure. The 81y cells were transduced at passage 6 with TRF1-citrine followed by transduction with lamin A. At passage 7, cells were immunolabeled with anti -H2AX antibody. Left panel shows merged images of lamin A-EGFP (blue) and TRF1-citrine (yellow), middle panel shows merged images of lamin A-EGFP (blue) and -H2AX (red), and right panel shows merged images of TRF1-citrine (yellow) and -H2AX (red) in normal (i) or senescent (ii,iii) cells.