Figure 8.
Figure 8. Inhibition of NPM1 blocks apoptosis and caspase-2–dependent suppression of cell growth. (A–C) Litter-matched Casp2+/+ and Casp2−/− MEFs (A), Raidd+/+ and Raidd−/− MEFs (B), or Pidd+/+ and Pidd−/− MEFs (C) were treated with or without camptothecin (250 nM). Apoptosis was assessed by flow cytometry for Annexin V binding 16 h later. Results are the mean of three to four independent experiments ± SD. *, P < 0.05. (D) Tp53−/− MEFs of indicated Npm1 genotypes were treated with or without camptothecin. Apoptosis was assessed by flow cytometry for Annexin V binding 16 h after treatment. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005. (E) Tp53−/− MEFs of indicated Npm1 genotypes were treated with or without Gö6976 (1 µM) ± IR (10 Gy), harvested 24 h after IR, stained for TUNEL, and analyzed by flow cytometry. Results are the mean of three independent experiments ± SD. *, P < 0.05. (F) HeLa cells transfected with the indicated siRNAs were treated with or without Gö6976 (1 µM) ± IR (10 Gy) and stained with alamarBlue 72 h after IR. Results are the mean of three independent experiments ± SD.*, P < 0.05 (two-tailed Student’s t test). (G–J′) p53M214K/M214K zebrafish embryos were injected at the one-cell stage with standard control (ctrl) or npm1a+npm1b morpholinos, incubated 17 h later with or without Gö6976 at indicated concentrations (μM), treated with or without 15 Gy IR, and stained with the cell death marker acridine orange (AO) after 7 h. All embryos were imaged live at 24 h postfertilization. Panels G′, H′, I′, and J′ are blowups of indicated spinal cord areas in corresponding whole-embryo panels. Bars, 250 µm. (K) Quantification of AO stains shown in G–J′. Results are the mean of three independent experiments (≥10 embryos per condition) ± SEM. **, P < 0.005. (L) Litter-matched Casp2+/+ and Casp2−/− MEFs, plated at low density, were treated with or without a chemical inhibitor of NPM1 (3 µM) for 7 h. Colonies were stained with methylene blue 5 d after treatment. The number of colonies under each treatment condition is shown. Results are the mean of six individual wells across two independent experiments ± SD. **, P < 0.005; ***, P < 0.0005. (M) Representative images of methylene blue–stained colonies from cells treated as in L.

Inhibition of NPM1 blocks apoptosis and caspase-2–dependent suppression of cell growth. (A–C) Litter-matched Casp2+/+ and Casp2−/− MEFs (A), Raidd+/+ and Raidd−/− MEFs (B), or Pidd+/+ and Pidd−/− MEFs (C) were treated with or without camptothecin (250 nM). Apoptosis was assessed by flow cytometry for Annexin V binding 16 h later. Results are the mean of three to four independent experiments ± SD. *, P < 0.05. (D) Tp53−/− MEFs of indicated Npm1 genotypes were treated with or without camptothecin. Apoptosis was assessed by flow cytometry for Annexin V binding 16 h after treatment. Results are the mean of three independent experiments ± SD. *, P < 0.05; **, P < 0.005. (E) Tp53−/− MEFs of indicated Npm1 genotypes were treated with or without Gö6976 (1 µM) ± IR (10 Gy), harvested 24 h after IR, stained for TUNEL, and analyzed by flow cytometry. Results are the mean of three independent experiments ± SD. *, P < 0.05. (F) HeLa cells transfected with the indicated siRNAs were treated with or without Gö6976 (1 µM) ± IR (10 Gy) and stained with alamarBlue 72 h after IR. Results are the mean of three independent experiments ± SD.*, P < 0.05 (two-tailed Student’s t test). (G–J′) p53M214K/M214K zebrafish embryos were injected at the one-cell stage with standard control (ctrl) or npm1a+npm1b morpholinos, incubated 17 h later with or without Gö6976 at indicated concentrations (μM), treated with or without 15 Gy IR, and stained with the cell death marker acridine orange (AO) after 7 h. All embryos were imaged live at 24 h postfertilization. Panels G′, H′, I′, and J′ are blowups of indicated spinal cord areas in corresponding whole-embryo panels. Bars, 250 µm. (K) Quantification of AO stains shown in G–J′. Results are the mean of three independent experiments (≥10 embryos per condition) ± SEM. **, P < 0.005. (L) Litter-matched Casp2+/+ and Casp2−/− MEFs, plated at low density, were treated with or without a chemical inhibitor of NPM1 (3 µM) for 7 h. Colonies were stained with methylene blue 5 d after treatment. The number of colonies under each treatment condition is shown. Results are the mean of six individual wells across two independent experiments ± SD. **, P < 0.005; ***, P < 0.0005. (M) Representative images of methylene blue–stained colonies from cells treated as in L.

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