Figure 5.
Age-related reduction of XRCC4, Lig4 and Lig3 expression causes the decline of NHEJ capacity. (a) Western blot analysis of c-NHEJ factors in the two groups of cells. Cells were collected for the whole-cell lysate extraction and western blot analysis at 48 h post splitting. (b) Statistical comparison of XRCC4 and Lig4 expression between the two groups. Western blot results were further analyzed with ImageJ software (Bethesda, MD, USA) for quantification. (c) XRCC4 and Lig4 were successfully depleted in the 17-year-old cell line using siRNA transfections. Cells were transfected with siRNA twice with two days interval. On day 3 post the second siRNA transfection, cells were collected for protein extraction and western blot analysis. (d) No significant effect on NHEJ efficiency was observed with the two proteins depleted. On day 2 post the second siRNA transfection, 3 × 105 cells were transfected with 0.2 μg I-SceI linearized NHEJ reporter and 0.005 μg pDsRed2-N1. At 72 h post transfection, cells were collected for the FACS analysis. Error bars, S.D. (e) Depleting both proteins using siRNA causes a reduction of NHEJ fidelity. Similar to the analysis in (d), cells depleted with the indicated proteins using siRNA were further transfected with 0.2 μg I-SceI linearized NHEJ reporter, followed by DNA extraction and plasmid rescue. The repaired NHEJ constructs were further sequenced at the junction area. At least 20 clones with deletions were sequenced and quantified. (f) Western blot analysis of alt-NHEJ factors in the two groups of cells. (g) Statistical comparison of Lig3 expression between the two groups. (h) Western blot analysis of XRCC4 and Lig4 overexpression in the 63-year-old cell line. Cells at the amount of 1 × 106 were transfected with 5 μg XRCC4 or Lig4 expression vector. At 24 h post transfection, cells were collected for protein extraction and western blot analysis. (i) Simultaneous overexpression of XRCC4 and Lig4 significantly elevates NHEJ efficiency. Together with 5 μg the control vector, 2.5 μg XRCC4+2.5 μg control vector, 2.5 μg Lig4+2.5 μg control vector or 2.5 μg XRCC4+2.5 μg Lig4 vector, cells were transfected with 5 μg I-SceI vector and 0.015 μg pDsRed2-N1 vector. At 72 h cells were collected for analysis of NHEJ efficiency. All experiments were repeated at least six times. Error bars, S.D. (j) Overexpression of both XRCC4 and Lig4 rescues the decline of NHEJ fidelity in the 63-year-old cell line. Similar to the experiments performed in (i), at 72 h post co-transfection, cells were collected for DNA extraction and plasmid rescue. For each group, at least 20 clones with deletions were sequenced and further analyzed