Figure 1.
Figure 1. DNA-damaging agents induce caspase-2 BiFC in the nucleolus. (A) HeLa cells transfected with C2-Pro VC (20 ng), C2-Pro VN (20 ng), and dsRed-mito as a transfection reporter (10 ng) were treated with camptothecin (100 µM), irinotecan (100 µM), topotecan (100 µM), etoposide (100 µM), actinomycin D (1 µM), taxol (10 µg/ml), or vincristine (10 µM) plus qVD-OPH (20 µM) to prevent cell detachment caused by apoptosis. Cells were assessed for the percentage of dsRed-positive transfected cells that were Venus+ at 24 h, determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005 compared with untreated sample. (B) Schematic representation of caspase-2 structure and BiFC constructs. Constructs containing the caspase-2 prodomain (C2 Pro) are shown, each fused to the C- or N-terminal fragment of Venus. The bicistronic construct consists of C2 Pro-VC and C2 Pro-VN linked by a 2A self-cleaving peptide. (C) HeLa cells stably expressing C2 Pro-VC-2A-C2 Pro-VN-2A-mCherry (HeLa.C2 Pro-BiFC; parent) were single-cell cloned (clone), and each was treated with the indicated drugs (100 µM) plus qVD-OPH (20 µM). After 24 and 48 h, the percentage of mCherry-positive cells that were Venus+ was determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 compared with the untreated sample in each group. (D) HeLa.C2 Pro-BiFC cells transfected with fibrillarin-CFP were treated with camptothecin (100 µM), irinotecan (100 µM), topotecan (100 µM), etoposide (100 µM), or vincristine (10 µM) plus qVD-OPH (20 µM) for 24 h. Representative images show cells (red) with caspase-2 BiFC (yellow) in the nucleolus (blue) or cytoplasm after treatment. Bars, 10 µm. (E) Percentage of cells treated as in D that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05 determined from the percentage of nucleolar Venus-positive cells compared with untreated. (F) HeLa.C2 Pro-BiFC cells were treated with or without the Chk1 inhibitor, Gö6976 (0.5 µM, 1 µM), ± IR (10 Gy) plus qVD-OPH (20 µM). The percentage of cells that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined at 24 h from at least 50 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05 determined from the percentage of nucleolar Venus-positive cells compared with untreated. (G) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in cells treated as in F. Bars, 10 µm.

DNA-damaging agents induce caspase-2 BiFC in the nucleolus. (A) HeLa cells transfected with C2-Pro VC (20 ng), C2-Pro VN (20 ng), and dsRed-mito as a transfection reporter (10 ng) were treated with camptothecin (100 µM), irinotecan (100 µM), topotecan (100 µM), etoposide (100 µM), actinomycin D (1 µM), taxol (10 µg/ml), or vincristine (10 µM) plus qVD-OPH (20 µM) to prevent cell detachment caused by apoptosis. Cells were assessed for the percentage of dsRed-positive transfected cells that were Venus+ at 24 h, determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005 compared with untreated sample. (B) Schematic representation of caspase-2 structure and BiFC constructs. Constructs containing the caspase-2 prodomain (C2 Pro) are shown, each fused to the C- or N-terminal fragment of Venus. The bicistronic construct consists of C2 Pro-VC and C2 Pro-VN linked by a 2A self-cleaving peptide. (C) HeLa cells stably expressing C2 Pro-VC-2A-C2 Pro-VN-2A-mCherry (HeLa.C2 Pro-BiFC; parent) were single-cell cloned (clone), and each was treated with the indicated drugs (100 µM) plus qVD-OPH (20 µM). After 24 and 48 h, the percentage of mCherry-positive cells that were Venus+ was determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 compared with the untreated sample in each group. (D) HeLa.C2 Pro-BiFC cells transfected with fibrillarin-CFP were treated with camptothecin (100 µM), irinotecan (100 µM), topotecan (100 µM), etoposide (100 µM), or vincristine (10 µM) plus qVD-OPH (20 µM) for 24 h. Representative images show cells (red) with caspase-2 BiFC (yellow) in the nucleolus (blue) or cytoplasm after treatment. Bars, 10 µm. (E) Percentage of cells treated as in D that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined from at least 30 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05 determined from the percentage of nucleolar Venus-positive cells compared with untreated. (F) HeLa.C2 Pro-BiFC cells were treated with or without the Chk1 inhibitor, Gö6976 (0.5 µM, 1 µM), ± IR (10 Gy) plus qVD-OPH (20 µM). The percentage of cells that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined at 24 h from at least 50 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05 determined from the percentage of nucleolar Venus-positive cells compared with untreated. (G) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in cells treated as in F. Bars, 10 µm.

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