Caspase-2 activation in the nucleolus requires the NLS and is a response to DNA breaks. (A) 3D reconstructions of HeLa.C2 Pro-BiFC cells transfected with Histone H2A-mTurquoise or Actin-mTurquoise and treated with camptothecin (100 µM) plus qVD-OPH (20 µM) were composed from 0.2-µm serial confocal images through the z-plane of the cell. Representative orthogonal-slice views show cells (red) with caspase-2 BiFC (yellow) and the indicated coexpressed proteins (blue). The middle, right, and top panels are the xy, yz, and xz planes, respectively. The yz and xz planes intersect according to the crosshairs. Bars, 10 µm. (B) HeLa cells transfected with the C2-Pro (1–147) BiFC or the C2-CARD (1–109) BiFC plasmid pair (25, 50, or 100 ng of each) with Histone H2B RFP (50 ng) were treated with or without camptothecin (100 µM). Cells were assessed 24 h later for the percentage of RFP-positive transfected cells that were Venus+ in the nucleolus, nucleus, or cytoplasm determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005 determined from the percentage of Venus-positive cells in the nucleolus. (C) Representative confocal images show caspase-2 BiFC (yellow) and nuclear Histone H2B expression (red) in cells treated as in B. Bars, 20 µm. (D) HeLa.C2 Pro-BiFC cells transfected with 53BP1-TFP (2 µg) were treated with the indicated drugs (100 µM) plus qVD-OPH (20 µM). TFP-positive cells were counted for absence or presence of 53BP1 foci and the percentage of cells that were Venus+ in the nucleolus, nucleus, or cytoplasm. Results are the mean of three independent experiments ± SD. The association of cells with caspase-2 BiFC in the nucleolus and 53BP1 foci was significant across experiments (camptothecin, P = 7.58 × 10−6; topotecan, 3.06 × 10−7; etoposide, 1.61 × 10−3) as calculated by the Cochran–Mantel–Haenszel test. (E) Representative confocal images of cells treated as in D show 53BP1 (blue) and caspase-2 BiFC (yellow). Bars, 5 µm.
