Figure 5.
Figure 5. RAIDD is required for caspase-2 BiFC, but PIDD is required for caspase-2 BiFC only in the nucleolus. (A) Litter-matched Raidd+/− or Raidd−/− MEFs stably expressing C2 Pro-BiFC were treated with camptothecin (250 nM), irinotecan (250 nM), topotecan (250 nM), etoposide (250 nM), or vincristine (25 nM) 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 30 microscopy images per well. Results are the mean of three independent experiments ± SD. The differences in total Venus-positive cells between the Raidd+/− and Raidd−/− groups is significant to P = 0.0034. (B) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in Raidd+/− and Raidd−/− C2 Pro-BiFC MEFs. Bars, 5 µm. Arrowheads indicate locations of nucleoli that are magnified in the bottom panel (bars, 1 µm). See Fig. S3 C for full dataset images. (C) Litter-matched Pidd+/+ or Pidd−/− MEFs stably expressing C2 Pro-BiFC were treated with camptothecin (250 nM), irinotecan (250 nM), topotecan (250 nM), etoposide (250 nM), or vincristine (25 nM) 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 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 nucleolar Venus-positive cells between Pidd+/+ and Pidd−/− cells for each treatment. (D) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in Pidd+/+ and Pidd−/− C2 Pro-BiFC MEFs. Bars, 5 µm. Arrowheads indicate locations of nucleoli that are magnified in the lower panel (bars, 1 µm). See Fig. S3D for full dataset images. (E) PIDD or RAIDD was deleted from HeLa.C2 Pro-BiFC cells with CRISPR/Cas9 using two independent sgRNAs per gene. The percentage of cells treated with or without camptothecin (100 µM) plus qVD-OPH (20 µM) that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined after 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 the control treated group. The difference in total Venus-positive cells compared with camptothecin-treated control for ΔRAIDD cells is significant to P < 0.0001 (ΔRAIDD(57)) and P = 0.0187 (ΔRAIDD(76)). (F) Pidd+/+ and Pidd−/− C2-Pro BiFC MEFs were treated with or without Gö6976 (0.125, 0.25, or 0.5 µM) ± IR (10 Gy) plus qVD-OPH (20 µM). The percentage of cells that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined 24 h after treatment from at least 50 microscopy images per well. Results represent triplicate counts ± SD. *, P < 0.05; **, P < 0.005 determined from the percentage of Venus-positive cells in the nucleolus between Pidd+/+ and Pidd−/− cells for each treatment.

RAIDD is required for caspase-2 BiFC, but PIDD is required for caspase-2 BiFC only in the nucleolus. (A) Litter-matched Raidd+/− or Raidd−/− MEFs stably expressing C2 Pro-BiFC were treated with camptothecin (250 nM), irinotecan (250 nM), topotecan (250 nM), etoposide (250 nM), or vincristine (25 nM) 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 30 microscopy images per well. Results are the mean of three independent experiments ± SD. The differences in total Venus-positive cells between the Raidd+/− and Raidd−/− groups is significant to P = 0.0034. (B) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in Raidd+/− and Raidd−/− C2 Pro-BiFC MEFs. Bars, 5 µm. Arrowheads indicate locations of nucleoli that are magnified in the bottom panel (bars, 1 µm). See Fig. S3 C for full dataset images. (C) Litter-matched Pidd+/+ or Pidd−/− MEFs stably expressing C2 Pro-BiFC were treated with camptothecin (250 nM), irinotecan (250 nM), topotecan (250 nM), etoposide (250 nM), or vincristine (25 nM) 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 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 nucleolar Venus-positive cells between Pidd+/+ and Pidd−/− cells for each treatment. (D) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) in Pidd+/+ and Pidd−/− C2 Pro-BiFC MEFs. Bars, 5 µm. Arrowheads indicate locations of nucleoli that are magnified in the lower panel (bars, 1 µm). See Fig. S3D for full dataset images. (E) PIDD or RAIDD was deleted from HeLa.C2 Pro-BiFC cells with CRISPR/Cas9 using two independent sgRNAs per gene. The percentage of cells treated with or without camptothecin (100 µM) plus qVD-OPH (20 µM) that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined after 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 the control treated group. The difference in total Venus-positive cells compared with camptothecin-treated control for ΔRAIDD cells is significant to P < 0.0001 (ΔRAIDD(57)) and P = 0.0187 (ΔRAIDD(76)). (F) Pidd+/+ and Pidd−/− C2-Pro BiFC MEFs were treated with or without Gö6976 (0.125, 0.25, or 0.5 µM) ± IR (10 Gy) plus qVD-OPH (20 µM). The percentage of cells that were Venus+ in the nucleolus, nucleus, or cytoplasm was determined 24 h after treatment from at least 50 microscopy images per well. Results represent triplicate counts ± SD. *, P < 0.05; **, P < 0.005 determined from the percentage of Venus-positive cells in the nucleolus between Pidd+/+ and Pidd−/− cells for each treatment.

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