the bh3-only protein bid is dispensable bh3-only protein bid is dispensable ... analysis from the...

T Kaufmann et al

Cell, Volume 129

Supplemental Data

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,4 Ricky W. Johnstone, Vishva M. Dixit, and Andreas Strasser

T Kaufmann et al

Figure S1. Bid-Deficient Mice Respond Normally to Full-Body -Irradiation Bid-/- mice and wt littermates of matching sex were subjected to a single dose of -radiation (2.5 Gy) and sacrificed for analysis 20 h later. (A) Bid-deficient mice had comparable total body and organ weights (spleen and thymus) as wt controls both before and after exposure to irradiation. (B) Total numbers of leukocytes and lymphocytes in

T Kaufmann et al

peripheral blood did not differ between bid-/- and wt mice. (C) Cell subset composition analysis in thymus, spleen and lymph nodes from untreated and irradiated wt and bid-/-

mice. No difference in percentages of lymphocyte subsets was observed between bid-/-

and wt mice. (D) Total cellularity and cell subset composition of thymus and spleen were found to be comparable between both untreated and irradiated bid-/- and wt mice. All graphs represent means +/-SD from 3-6 independent mice per genotype and treatment.

Figure S2. Blood Counts of Aged Bid-Deficient Mice on an Inbred C57BL/6 Genetic

Background

(A) White blood cell counts from 21 of our Bid-deficient mice (aged 14-21 months) and 8 of the original Bid-deficient (bidneo/neo) mice (aged 18-24 months) do not show abnormally elevated leukocyte numbers as compared to our 6-12 week old Bid-deficient mice or C57BL/6 wt mice. Each dot represents a single animal. (B) Peripheral blood analysis from the same aged Bid-deficient mice as in (A) shows normal lymphocyte, neutrophil and platelet counts, as well as normal levels of hemoglobin (HGB). Data represent means +/-SD from the indicated number (n) of animals.

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Figure S3. Bid-Deficient Lymphocyte Sub-Populations Are Normally Sensitive to Dexamethasone, FasL, PMA and Ionomycin

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FACS-sorted lymphocyte sub-populations (CD4+8+ immature thymocytes, CD4+8- and CD4-8+ mature T cells from lymph nodes, B220+sIgMhiDlo immature B cells and B220+sIgMloDhi mature B cells from lymph nodes and B220+sIg-c-Kit- pre-B cells from bone marrow) of bid-/- and control (wt) littermates were treated with the indicated doses of dexamethasone, recombinant FasL (FLAG-tagged and crosslinked with anti-FLAG antibodies), PMA or ionomycin. 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.

T Kaufmann et al

T Kaufmann et al

Figure S4. Bid-Deficient Primary MEF Undergo Normal Cell Cycle Arrest after Exposure to DNA Damage or Replicative Stress Independent lines of early passage (P1) wt and bid-/- (A) or wt and bidneo/neo (derived from the originally described Bid-deficient mice) (B) primary MEF were left untreated, -irradiated ( -rad., 5 and 20 Gy) or treated for 2 h with the indicated doses of etoposide (etop., 10 g/ml = 17 M), mitomycin C (mito C, 1 M), hydroxyurea (HU, 1 mM) or aphidicolin (aphid., 5 or 10 M) followed by 8 h incubation in drug free medium. Cell cycle distribution was then analyzed as described in Figure 5A. Data represent means +/-SD using 3 independent primary MEF (passage 1) derived from 3 independent embryos of each genotype.