Figure 1

Visualization of telomeres in live cultured cancer cells. (A) A schematic of lentivector expressing green fluorescent protein (GFP)-tagged TRF1 or TRF2. The vector map is referenced in the Methods section. (B) Western blotting analysis of UMUC3 cells stably expressing GFP-TRF1 or GFP-TRF2 using antibodies against TRF1 or TRF2. Clonal lines with the lowest average expression (indicated by red numbers) were chosen for comparison with the endogenous level of TRF1 or TRF2 for telomere dynamic analysis. (C) Indirect immunofluorescence of clonal UMUC3 cell lines expressing either GFP-TRF1 (upper row) or GFP-TRF2 (lower row). Complete colocalization was observed from GFP-TRF1-expressed telomeres stained with anti-TRF2 antibody in fixed cells in the upper row. Lower row: immunofluorescence images of GFP-TRF2-expressed telomeres stained with anti-hRap1 antibody also showed telomere colocalization. (D) A schematic of the data collection regime for a representative UMUC3 nucleus. One complete three-dimensional (3-D) stack of 8 μm deep (an entire UMUC3 nucleus) with 0.5 μm focal spacing (17 sections) was acquired every second. (E) Representative examples of projection images from one complete 3-D stack of GFP-TRF1-expressed UMUC3 nucleus that was acquired every second for 200 consecutive seconds (T, time in seconds). The photobleaching curve is shown in D in Additional file 1.