MazF inhibits protein synthesis through degradation of cellular mRNAs in mammalian cells. (A) Northern blot analysis of human GAPDH and β-actin. Total RNA from Tet-treated or -untreated T-Rex-293 cells at the indicated time points was probed with ³²P-labeled human GAPDH and β-actin cDNA. 28S and 18S ribosomal RNAs were visualized by agarose-formaldehyde gel electrophoresis followed by ethidium bromide staining. (B) Quantification of mRNA levels. Human GAPDH and β-actin mRNA levels were quantified by real-time RT–PCR. Relative amounts of mRNA were calculated from the fluorescence signal in the 24-, 48-, and 72-h samples as compared with the corresponding 0-h sample. (C) ³⁵S-methionine incorporation in T-Rex-293 cells. ³⁵S-methionine-labeled total protein from Tet-treated T-Rex-293 cells at the indicated time points was subjected to SDS-PAGE and autoradiography (left) and stained with Coomassie blue (right). (D) Quantification of ³⁵S-methionine-labeled proteins. Protein bands on the gel in C were scanned by PhosphorImager Storm 860 (Molecular Dynamics) and signal intensity was calculated.

Levels of BCL-2 family proteins remain constant during MazF induction. Whole-cell lysates from Tet-treated T-Rex-293 cells were immunoblotted with antibodies that specifically recognize the anti-apoptotic and proapoptotic proteins indicated in the figure.

NBK/BIK mediates MazF or CHXinduced cell death. (A) Viability of iBMK cells transiently expressing MazF. nbk/bik^−/−, bim^−/−, noxa^−/−, or puma^−/− iBMK cells coexpressing LacZ and MazF were subjected to a β-galactosidase assay. β-Galactosidase-positive blue cells were calculated as a percentage of total cells. Data are representative of three independent experiments. (B,C) Viability of CHX-treated iBMK cells. W2, D3, X2, and K1 cells (B), and nbk/bik ^−/−B, bim^−/−, noxa^−/−, or puma^−/− cells (C) treated with CHX were subjected to an MTT assay. Data are representative of three independent experiments. (D,E) Viability of TNF-α/CHX- and paclitaxel-treated iBMK cells. W2, D3, and three independent nbk/bik^−/− cell lines (A, B, and C) treated with TNF-α/CHX (0.05 μg/mL) (D) and paclitaxel (E) were subjected to an MTT assay. Data are representative of three independent experiments.

NBK/BIK mediates adenovirus-induced apoptosis. (A, top) Phase-contrast photographs of adenovirus-infected iBMK cells (magnification 100×). (Bottom) Adherent cell number was presented with error bars by counting adherent cells in three different images. (B) Western blot analysis with lysates from viral-infected iBMK cells. Whole-cell lysates from mock-, Ad5dl309-, or Ad5dl337-infected W2, D3, and nbk/bik ^−/− B cells were immunoblotted with an anti-active caspase-3 antibody (top), anti-E1A antibody (middle), and anti-actin antibody (bottom). (C) Apoptosis pathway induced by shutoff of protein synthesis. See text for details.

MazF induces apoptotic cell death in mammalian cells. (A) Identical frames from 4-d time-lapse videos of Tet-treated or -untreated T-Rex-293 cells performed as described previously (Degenhardt et al. 2006). (B) Viability analysis of T-Rex-293 cells in A. Tet-treated or -untreated T-Rex-293 cells at the indicated time points were subjected to trypan blue exclusion. Viability from representatives of three independent experiments was presented as a percent of total cells at time 0. (C) Representative illustration of propidium iodide labeling measured by FACS in Tet-treated T-Rex-293 cells. (D) Western blot analysis with lysates from T-Rex-293 cells. Whole-cell lysates from Tet-treated or staurosporine-treated T-Rex-293 cells at the indicated time points were immunoblotted with an anti-active caspase-3 antibody (top), anti-PARP antibody (middle), and anti-actin antibody (bottom).

BAK function is required for MazF-induced apoptosis. (A) Viability of iBMK cells transiently expressing MazF. W2, D3, X2, and K1 cells transiently coexpressing LacZ and MazF were subjected to a β-galactosidase assay at 48 h post-transfection. β-Galactosidase-positive blue cells were calculated as a percentage of total cells. Data are representative of three independent experiments. (B) Immunofluorescence of activated caspase-3 in iBMK cells. W2, D3, X2, or K1 cells transiently coexpressing LacZ and MazF were costained with anti-Xpress and anti-active caspase-3 antibody. FITC (green) and Rhodamine (red) stains represent cells expressing LacZ and activated caspase-3, respectively. Numbers represent the percentage of activated caspase-3-positive cells. White arrows indicate the corresponding activated caspase-3-positive cells from the matching FITC-stained cells. (C) Viability of iBMK cells transiently coexpressing MazF and MazE. W2, D3, X2, and K1 cells transiently coexpressing LacZ and MazF and/or MazE were subjected to a β-galactosidase assay. β-Galactosidase-positive blue cells were calculated as described above. Data are representative of three independent experiments. (D) ³⁵S-methionine incorporation upon MazF induction in BAX/BAK-deficient D3 cells. ³⁵S-methionine-labeled total protein from Tet-treated D3/mazF and D3 cells at the indicated time points was subjected to SDS-PAGE and autoradiography (left) and stained with Coomassie blue (right). Note the pronounced inhibition of protein synthesis when MazF is expressed. (E) Viability analysis of D3/mazF and D3 cells. Tet-treated or -untreated D3/mazF and D3 cells at the indicated time points were subjected to trypan blue exclusion. Viability was presented with error bars as a percent of total cells. Despite the profound inhibition of ³⁵S-methionine incorporation upon MazF induction (shown in D), defective apoptosis conferred by BAX/BAK deficiency maintained cell viability.

NBK/BIK interacts MCL-1 and BCL-X_L to activate BAK in MazF- and CHX-induced apoptosis. (A) Immunoprecipitation (IP) with lysates from 293 cells transiently expressing Myc-NBK/BIK. Immunoprecipitation of MCl-1 and Myc-NBK/BIK, BCL-X_L, and Myc-NBK/BIK from the soluble fraction of Tet-treated 293 cells transiently expressing Myc-NBK/BIK was carried out with anti-Myc antibody. Western blotting was carried out on precipitated samples and on lysates collected before immunoprecipitation with anti-MCL-1, anti-BCL-X_L or anti-Myc antibody. (B) Immunoprecipitation (IP) with lysates from Tet-treated or untreated 293 cells. Immunoprecipitation of BAK, MCL-1, and BCL-X_L from the soluble fraction of Tet-treated or -untreated 293 cells was carried out with anti-MCL-1 and anti-BCL-X_L antibodies. Western blotting was carried out on precipitated samples and on lysates collected before immunoprecipitation with anti-MCL-1, anti-BCL-X_L, and anti-BAK antibodies. (C,D) Immunoprecipitation (IP) with lysates from 1 μg/mL CHX-treated 293 cells transiently expressing Myc-NBK/BIK. Immunoprecipitation was carried out as indicated in A and B.