1. Volume 129, Issue 2, Pages 423-433 (April 2007)
The BH3-Only Protein Bid Is Dispensable for DNA Damage- and Replicative Stress- Induced Apoptosis or Cell-Cycle Arrest 
Thomas Kaufmann, Lin Tai, Paul G. Ekert, David C.S. Huang, Fiona Norris, Ralph K. Lindemann, Ricky W. Johnstone, Vishva M. Dixit, Andreas Strasser 
Cell 
Volume 129, Issue 2, Pages (April 2007)
DOI: /j.cell
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Generation of Bid-Deficient Mice and Demonstration that these Animals Are Resistant to Anti-Fas Antibody-Induced Fatal Hepatitis
(A) Schematic representation of the mouse bid locus, the targeting vector, and the knockout allele (abbreviations: H indicates HindIII, S indicates SacII, and N indicates NheI).
(B) Southern blot analysis of restriction-enzyme-digested genomic DNA from selected electroporated C57BL/6-derived ES cell clones.
(C) Three-primer PCR analysis of genomic DNA to determine mouse genotypes.
(D) Western blot analysis using a commercially available anti-Bid monoclonal antibody (BD Pharmingen) and proving the absence of detectable levels of Bid protein in extracts from thymocytes of bid−/− mice. Asterisk denotes Ig light chain. As a loading control, the membrane was reprobed with an antibody to β-actin.
(E) Western blot analysis with two newly developed Bid-specific rat monoclonal antibodies (clones 2D1 and 8C3). Note the specificity of both antibodies for both full-length as well as truncated tBid. In order to induce Bid cleavage, total (unsorted) thymocytes or purified CD4+8+ thymocytes were incubated for 2 hr with recombinant FLAG-tagged FasL (100 ng/ml, crosslinked with anti-FLAG antibody; lanes 4 and 6). As a loading control, membranes were probed with an antibody to β-actin.
(F) Bid-deficient mice, WT littermate controls, and Fas mutant lpr mice were injected i.v. with either carrier (PBS) or a lethal dose (0.25 μg/g body weight) of agonistic anti-Fas antibody (clone Jo2). All mice were sacrificed after 200 min, then bled, and their sera were analyzed for the liver enzymes ALT and AST. Each circle represents a single mouse. Numbers (n) of mice analyzed are indicated.
(G) Histological examination of liver sections from WT or bid−/− mice injected 200 min earlier with Jo2 anti-Fas antibody (bars = 50 μm). Pictures shown are representative of the analysis of at least three mice for each treatment and genotype.
Cell  , DOI: ( /j.cell )
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3. Figure 2
Bid-Deficient Lymphocytes Are Normally Sensitive to DNA Damage-Induced Apoptosis In Vitro
FACS-sorted lymphocyte subsets (CD4+8+ immature thymocytes, CD4+8− and CD4−8+ mature T cells from lymph nodes, B220+sIg−c-Kit− pre-B cells from bone marrow, and B220+sIgMloDhi mature B cells from lymph nodes) of the newly generated bid−/− mice and WT littermates (A) or of the original Bid-deficient (bidneo/neo) mice (backcrossed for >10 generations onto the C57BL/6 background) and C57BL/6 WT control mice (B) were treated with the indicated doses of etoposide or γ-radiation. Percentages of viable cells were determined at the indicated time points by staining with PI plus AnnexinV-FITC followed by flow cytometric analysis. Data represent means ±SD from cells of 3–6 independent mice of each genotype.
Cell  , DOI: ( /j.cell )
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4. Figure 3
Bid Is Dispensable for DNA Damage- and Replicative Stress-Induced Apoptosis of Diverse Cell Types In Vitro
(A) bid−/− and WT activated T cells were deprived of IL-2, γ-irradiated, or treated with etoposide. Percentages of viable (PI-negative) cells were assessed as indicated in Figure 2. Data represent means ±SD of three independent experiments.
(B) E1a/ras-transformed MEF were exposed to etoposide, γ-radiation, or UVC-radiation, and percentages of viable (PI-negative) cells were assessed over time by flow cytometry. Data represent means ±SD of three independent WT and five independent bid−/− clones.
(C) Immortalized myeloid progenitor cells (FDM) generated from bid−/− or WT fetal-liver-derived hemopoietic progenitors were exposed to the indicated doses of etoposide, γ-radiation, or the replicative stress-inducing drugs aphidicolin, hydroxyurea, or mitomycin C. Cell viability was measured as indicated in Figure 2. Data represent means ±SD of three independent clones per genotype.
(D) The same cells as in (C) were treated for 24 hr with the indicated drugs at the same concentrations as in (C). Nuclear DNA was then stained with PI (using hypotonic buffer), and cells with a subG1 DNA content enumerated by flow cytometry (n = 5 for WT; n = 4 for bid−/−).
(E) bid−/− or WT FDM cells were exposed to mitomycin C or left untreated for 24 hr. The cells were arrested, stained, and scored for chromatid breaks and triradial figures. No radial figures were recorded (not shown). Twenty-five cells were analyzed per independent, clonal FDM cell line (n = 3 for WT; n = 4 for bid−/−). Data are represented as numbers of chromatid breaks per cell.
Cell  , DOI: ( /j.cell )
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5. Figure 4
Various Cell Types from the Originally Described Bid-Deficient Mice Are Normally Sensitive to DNA Damage- and Replicative Stress-Induced Apoptosis In Vitro
(A) Spleen cells from the original Bid-deficient (bidneo/neo) mice and C57BL/6 WT control animals were purified on a Ficoll gradient and then treated with the indicated doses of etoposide. Percentages of dead cells after 24 or 40 hr in culture were determined by PI uptake and flow cytometric analysis. Percentages of dead cells at t = 0 hr were 9.4 ± 0.6% for WT and 7.3 ± 0.4% for bid−/− mice, respectively. Data represent means ±SD of three mice of each genotype.
(B) Activated T cells from the original Bid-deficient (bidneo/neo) mice and C57BL/6 WT control animals were treated with the indicated doses of the DNA-damaging drug etoposide, γ-irradiated, or exposed to the replicative stress inducing drugs hydroxyurea or aphidicolin. Percentages of viable cells were determined at the indicated time points by staining with PI plus AnnexinV-FITC followed by flow cytometric analysis. Data were normalized to untreated control samples and represent means ±SD of three mice of each genotype.
(C) E1a/ras-transformed MEF derived from the original Bid-deficient (bidneo/neo) or control WT embryos were exposed to etoposide, γ-radiation, or hydroxyurea, and percentages of viable (PI negative) cells were assessed over time by flow cytometry. Data represent means ±SD of three independent clones (each clone derived from a different embryo) of each genotype.
(D) Western blot analysis using anti-Bid monoclonal antibodies confirms the absence of Bid in cells used in (C) from the original Bid-deficient (bidneo/neo) mice. Probing with an antibody to β-actin served as a loading control.
Cell  , DOI: ( /j.cell )
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6. Figure 5
Bid-Deficient Cells Undergo Normal Cell-Cycle Arrest after Exposure to DNA Damage or Replicative Stress
(A) Independent clones of WT and bid−/− or WT and bidneo/neo (derived from original Bid-deficient mice) E1a/ras-transformed MEF were left untreated, γ-irradiated (γ-rad., 5 Gy), or treated for 2 hr with etoposide (etop., 10 μg/ml = 17 μM), mitomycin C (mito C, 100 nM), aphidicolin (aphid., 5 μM), or hydroxyurea (HU, 100 μM) followed by 8 hr of culture in drug-free medium. The cells were then lysed in hypotonic PI buffer, and the cell-cycle profiles analyzed by flow cytometry.
(B) Independent clones of WT and bid−/− or WT and bidneo/neo (derived from original Bid-deficient mice) FDM were left untreated, γ-irradiated (γ-rad., 1.25 or 5 Gy), or treated for 24 hr with etoposide (etop., 1 μg/ml = 1.7 μM), aphidicolin (aphid., 0.1 μM), hydroxyurea (HU, 100 μM), or mitomycin C (mito C, 100 nM). Nuclear DNA was then stained with PI (using hypotonic buffer), and the cell cycle profiles were analyzed by flow cytometry. Data in (A) and (B) represent means ±SD of three independent clones (from three independent embryos) for each genotype.
Cell  , DOI: ( /j.cell )
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7. Figure 6
Loss of Bid Does Not Affect Clonogenic Death of γ-Irradiated MEF or Mitogen-Induced Proliferation of T Lymphocytes
(A) bid−/− or WT E1a/ras-transformed MEF were seeded at 1 × 103 cells per well (6-well plate) the day prior to γ-irradiation with the indicated doses. After 7 days, the cells were fixed and stained with crystal violet, and the number of colonies per well was counted. Data are presented as percentage of surviving colony-forming cells compared to the nonirradiated control cultures giving means ±SD of three independent clonal E1a/ras-transformed MEF (from three independent embryos) of each genotype. Only one well from a 6-well plate is shown in the illustrative photograph.
(B) Purified bid−/− and control (WT) T lymphocytes were activated with anti-CD3 antibody, anti-CD3 plus anti-CD28 antibodies, or ConA. The relative cell size (forward light scatter: FSC) of activated T cells was determined at the indicated time points in a FACScan and is represented as mean FSC. The viable number of activated T cells was measured by PI staining and flow cytometry using FACS reference beads. Data are represented as fold-increase of viable cells, giving means ±SD from T cells of three independent mice of each genotype.
Cell  , DOI: ( /j.cell )
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8. Figure 7
Bid Is a Cytoplasmic Protein and Does Not Translocate into the Nucleus after DNA Damage or Replicative Stress
E1a/ras-transformed MEF (A), FDM cells (B), or activated T cells (C; all from WT C57BL/6 mice) were left untreated or were treated for 1 hr with etoposide (etop., 100 μM = 58.8 μg/ml) or hydroxyurea (HU, 1 mM) or were γ-irradiated (γ-rad., 20 Gy) and then cultured for 1 hr. Nuclear (N) and cytoplasmic (C) fractions were prepared and equal amounts per fraction separated on SDS-PAGE gels, then western blotted and probed for Bid as well as for the cytosolic protein HSP70 and the nuclear protein PARP, both of which served as controls for the purity of the subcellular fractions.
Cell  , DOI: ( /j.cell )
Copyright © 2007 Elsevier Inc. Terms and Conditions