Ku levels in young and senescent cells. (A) Young and senescent IMR-90 and HCA2 cells were harvested and 30 μg of whole cell protein extract was analyzed by Western blot with Ku70 or Ku80 antibodies. Equal loading was confirmed by hybridization with actin, GAPDH, and Lamin B1 antibodies. Y, young cells; S, senescent cells. (B) The Western blots were quantified using ImageQuant software. Protein levels in senescent cells were normalized to the young cells, which were taken as 100%. Black bars represent young cells; white bars represent senescent cells. The experiments were repeated four times, and error bars show standard deviations.

Subcellular distribution of Ku70 and Ku80 in young and senescent cells. (A) Western blot analysis of Ku in subcellullar fractions. Young and senescent IMR-90 and HCA2 cells were fractionated into cytoplasmic (C), membrane/organelle (M), nuclear (N), and cytoskeletal (S) fractions. The fractions were loaded according to the percent of total cellular protein represented by each fraction. Fractions were verified by hybridizing with LDH, Na⁺/K⁺ ATPase, and histone H3 antibodies. (B) The Western blots were quantified using ImageQuant software. The fractions were plotted according to the percent of total cellular protein represented by each fraction. The sum of the three fractions in the young cells was taken as 100%; the sum of the three fractions in senescent cells was reduced according to the data in Figure 1. Black bars represent young cells; white bars represent senescent cells. Experiments were repeated four times, and error bars show standard deviations.

Immunohistochemical analysis of Ku localization in young and senescent human fibroblasts. Young and senescent IMR-90 and HCA2 cells were fixed and incubated with Ku80 antibodies as described in Materials and Methods. Green color is immunofluorescent staining of Ku80; blue color is DNA stained with DAPI. (A) Untreated young and senescent cells. (B) Young cells 24 hours after γ-irradiation. (C) Young cells 24 hours after UVC treatment.

Ku levels and intracellular localization in young arrested cells. (A) Young IMR-90 and HCA2 cells were kept at confluence for 7 days. The total levels of Ku were analyzed by Western blot in whole cell extracts. (B) Cells were kept at confluence for 48 hours or for 7 days and subcellular distribution of Ku was analyzed by cell fractionation as in Figure 2. C, cytoplasmic fraction; M, membrane/organelle fraction, N, nuclear fraction; and S, cytoskeletal fraction.

Subcellular distribution of Ku70 and Ku80 in young and senescent cells following exposure to γ-irradiation. IMR-90 and HCA2 cells were irradiated with 5 or 50 Gy of γ-irradiation, and fractionated. (A) Representative Western blots showing changes in subcellular localization of Ku70 or Ku80 after 50 Gy of γ-irradiation. The fractions were loaded according to the percent of total cellular protein represented by each fraction. C, cytoplasm; M, membranes/organelles, N, nucleus; S, cytoskeleton. (B) Quantification of the Western blots obtained after 50 Gy of γ-irradiation. Quantification was done using ImageQuant software. The results for IMR-90 and HCA2 cells were pooled. The fractions were plotted according to the percent of total cellular protein represented by each fraction. The sum of the three fractions in the young cells was taken as 100%; the sum of the three fractions in senescent cells was reduced according to the data in Figure 1. (C) Quantification of the Western blots obtained after 5 Gy of γ-irradiation. Bar's shading represent different time points after irradiation: black bars, 0 time point; dark grey bars, 1 hour after irradiation; light grey bars, 5 hours after irradiation; and white bars, 24 hours after irradiation. The experiments were repeated twice for each cell line and standard deviations are shown.

Subcellular distribution of Ku70 and Ku80 in young and senescent cells following UVC irradiation. IMR-90 and HCA2 cells were irradiated with 40×100 or 400×100 μJ/cm² of UVC and fractionated. (A) Representative Western blots showing changes in subcellular localization of Ku70 or Ku80 after 400×100μJ/cm² of UVC. The fractions were loaded according to the percent of the total cellular protein represented by each fraction. C, cytoplasm; M, membranes/organelles, N, nucleus; S, cytoskeleton. (B) Quantification of the Western blots obtained after the high dose (400×100μJ/cm²) of UVC. Quantification was done using ImageQuant software. The results for IMR-90 and HCA2 cells were pooled. The fractions were plotted according to the percent of total cellular protein represented by each fraction. The sum of the three fractions in the young cells was taken as 100%; the sum of the three fractions in senescent cells was reduced according to the data in Figure 1. (C) Quantification of the Western blots obtained after the low dose of UVC (40×100μJ/cm²). Bar's shading represent different time points after irradiation: black bars, 0 time point; dark grey bars, 1 hour after irradiation; light grey bars, 5 hours after irradiation; and white bars, 24 hours after irradiation. The experiments were repeated twice for each cell line and standard deviations are shown.

Senescence-related changes in Ku distribution and response to DNA damage. In the young cells Ku (open circles) is present predominantly in the nucleus and cytoplasm. In response to γ-irradiation Ku moves into the nucleus and membrane/organelle fraction. In response to UVC Ku moves out of the nucleus. In senescent cells the majority of Ku protein is localized in the nucleus, presumably in a DNA-bound form (crossed circles). Ku in senescent cells does not change its localization in response to γ-irradiation or UVC.