Figure 2. Timeless-depleted cells have telomere specific DNA damage. (A) Telomere dysfunction-induced foci (TIF) assay of MCF-10A cells expressing the indicated shRNA. Cells were in situ-extracted with Triton X-100. Colocalization of 53BP1 and TRF1 was determined using anti-53BP1 (red) and anti-TRF1 (green) antibodies. DNA was co-stained with DAPI (4’, 6’-diamidino-2-phenylindole). The merged images of 53BP1, TRF1 and DNA are shown. (B) Quantification of cells containing TIFs (colocalization of 53BP1 and TRF1). Cells with four or more TIFs were scored as TIF positive (n > 100 nuclei; Error bars correspond to standard deviations obtained from three independent experiments). *Denotes P-value determined by paired Student’s t-test.