www.sciencemag.org/cgi/content/full/330/6009/1390/DC1

Supporting Online Material for

BID, BIM, and PUMA Are Essential for Activation of the BAX- and BAK-Dependent Cell Death Program

Decheng Ren, Ho-Chou Tu, Hyungjin Kim, Gary X. Wang, Gregory R. Bean, Osamu Takeuchi, John R. Jeffers, Gerard P. Zambetti,

James J.-D. Hsieh, Emily H.-Y. Cheng*

*To whom correspondence should be addressed. E-mail: chenge1@mskcc.org

Published 3 December 2010, Science 330, 1390 (2010) DOI: 10.1126/science.1190217

This PDF file includes:

Materials and Methods Figs. S1 to S15 Tables S1 and S2 References

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MATERIALS AND METHODS

Generation of knockout Mice Bid-/-, Bim-/-, Puma-/-, Baxf/f, Nestin-Cre+ mice were described previously (1-4). To

generate Bid-/-Bim-/- mice, Bid+/-Bim+/- mice were crossed to Bid+/-Bim+/- mice, or Bid-/-

Bim+/- mice were crossed to Bid-/-Bim+/- mice. To generate Bid-/-Bim-/-Puma-/- mice, Bid+/-

Bim+/-Puma+/- mice were crossed with Bid+/-Bim+/-Puma+/- mice, Bid-/-Bim+/-Puma+/- mice

were crossed with Bid-/-Bim+/-Puma+/- mice, Bid+/-Bim+/-Puma-/- mice were crossed with

Bid+/-Bim+/-Puma-/- mice, or Bid-/-Bim+/-Puma-/- mice were crossed with Bid-/-Bim+/-Puma-/-

mice. Animal experiments were performed according to institutional guidelines.

Irradiation of Mice and Immunohistochemistry

Mice at postnatal day 5 (P5, day of birth is P0) were irradiated with 14 Gy γ-irradiation.

Brain tissues were collected at 18 or 30 hours post-irradiation and fixed in Bouin’s

solution (Ricca Chemical Company). Paraffin embedded brain tissues were sectioned at 7

µm in thickness, and subjected to immunohistochemistry against cleaved-caspase-3 (Cell

Signaling Technology). Antigen retrieval was performed in a Decloaking Chamber

(Biocare Medical) using Reveal Decloaker RTU buffer (Biocare Medical). Prior to

primary antibody incubation, the samples were sequentially blocked with Peroxidase

Blocking Ready-to-use Reagent (Dako) and DakoCytomation ready-to-use Protein Block

(Dako). The samples were then incubated with anti-cleaved caspase-3 antibody at a

dilution of 1:1,000, followed by biotinylated goat anti-rabbit-HRP (Dako) secondary

antibody at a dilution of 1:500. The Vectastain Elite ABC kit (Vector laboratories) and

DAB+ liquid (Dako) were used for visualization of the primary antibody.

Cerebellar granule neuron cultures and potassium deprivation assays

Primary cultures of cerebellar granule neuron (CGN) were performed as described

previously (5). Briefly, postnatal day 5 (P5) cerebella were dissected, trypsinized,

triturated, and plated into K25+S medium (glutamine-free Basal Media Eagle with

Earle’s Salts supplemented with 10% dialyzed fetal calf serum, 2 mM L-glutamine, 25

mM KCl, and 0.02 mg/ml gentamicin) at a density of 5×105 cells per well of 24-well

plates coated with poly-L-lysine. To reduce the number of non-neuronal cells, 3.3 µg/ml

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aphidicolin was added to the medium 24 hours after initial plating. The culture conditions

do not support the survival of other neuronal cell types and the non-neuronal

contamination was less than 2%. On the 7th day of culture, CGN were either maintained

in K25+S or switched to K5+S medium (glutamine-free Basal Media Eagle with Earle’s

Salts supplemented with 10% dialyzed fetal calf serum, 2 mM L-glutamine, 5 mM KCl,

and 0.02 mg/ml gentamicin) after washing once with the respective medium. Cell

viability was then assessed at the indicated times by taking photomicrographs of

representative fields of neurons stained with propidium iodide (0.5 µg/ml).

Cell culture and viability assays

Thymocytes were isolated from mice at 6-8 weeks of age. Single cell suspensions of

thymocytes were prepared and cell numbers were determine by HEMAVET (Drew

Scientific). Splenocytes were isolated from mice at 8-10 weeks of age. Single-cell

suspensions of splenocytes were prepared and cell numbers were determined by

HEMAVET (Drew Scientific). CD4+ T cells were isolated using anti-CD4 conjugated

magnetic beads (Miltenyi). Thymocytes or CD4+ T cells were cultured in complete

RPMI-1640 with 10% fetal bovine serum (Invitrogen). Apoptosis was induced by 10 nM

dexamethasone (Sigma), 1 µg/ml tunicamycin (Sigma), 1 µM etoposide (Sigma), 2.5 Gy

γ-irradiation, or 1 µg/ml anti-Fas antibody (clone Jo2, BD Biosciences). Primary mouse

embryonic fibroblasts (MEFs) were generated from E13.5 embryos and cultured in

Iscove's Modified Dulbecco's Medium supplemented with 20% fetal bovine serum

(Invitrogen). All MEFs used for experiments were within 2 passages. Retroviruses

expressing various BH3-only proteins were described previously (8). Cell death was

quantified by annexin-V (BioVision) or propidium iodide (Sigma) staining, followed by

flow cytometric analyses. Flow cytometry was performed using a FACSCalibur (BD

Biosciences), and data analyzed using CellQuest Pro software (BD Biosciences) or FloJo

software. P values for statistical analyses were obtained using Student’s t test.

Cytochrome c release assay Mitochondria purification and cytochrome c release assays were performed as described

previously (6-8). Quantification of cytochrome c release was performed using

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colorimetric ELISA assays (MCTC0, R&D Systems). To obtain the percentage of

cytochrome c release, the amount of cytochrome c present in mitochondrial supernatant

was divided by total cytochrome c present in both mitochondrial supernatant and pellet.

Gel filtration chromatography

The chromatographic step of Superdex 200 (HR 10/30, GE-Amersham) was performed

on an automatic fast protein liquid chromatography (AKTApurifier, GE-Amersham). The

column was equilibrated with 2% CHAPS buffer (2% CHAPS, 300 mM NaCl, 0.2 mM

DTT, 20 mM HEPES pH 7.5) and calibrated with thyroglobulin (669 kD, GE-

Amersham), ferritin (440 kD, GE-Amersham), catalase (232 kD, GE-Amersham),

aldolase (158 kD, GE-Amersham), bovine serum albumin (66 kD, Calbiochem), and

cytochrome c (14 kD, Sigma). Thymocytes were lysed in 2 % CHAPS buffer

supplemented with complete protease inhibitor (Roche). Protein concentration was

determined by BCA kit (Pierce). 200 µl of lysates (2 mg/ml) were loaded onto the

column, eluted at a flow rate of 0.3 ml/min. Fractions of 0.6 ml were collected,

precipitated by trichloroacetic acid, and analyzed by 8-16% SDS-PAGE (Bio-Rad) and

anti-BAX or anti-BAK immunoblots.

Immunoblots and indirect immunofluorescence microscopy

Cells were lysed in PBS containing 1% Triton-X 100. Protein concentration was

determined by BCA kit (Pierce). 25-50 µg of proteins were resolved by 10% NuPAGE

(Invitrogen), transferred onto PVDF membrane (Immobilon-P, Millipore). Antibody

detection was accomplished using enhanced chemiluminescence method (Western

Lightning, PerkinElmer) and LAS-3000 Imaging system (FUJIFILM). Antibodies used

for Western Blots are listed as followed: anti-BAK (NT, Upstate), anti-BAX (N-20, Santa

Cruz), anti-BIM (Calbiochem), anti-PUMA (Sigma), anti-BAD (C-20, Santa Cruz), anti-

BMF (17A9, Alexis), anti-BCL-2 (3F11), anti-BCL-XL (Cell Signaling Technology),

anti-MCL-1 (Santa Cruz), anti-BCL-W (Calbiochem), anti-A1 (R&D Systems), anti-

cleaved caspase-3 (Cell Signaling Technology), anti-PARP (Cell Signaling Technology),

and anti-actin (Chemicon).

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Cells, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100,

were sequentially incubated with anti-cytochrome c antibody (BD Biosciences), Alexa

Fluor 568 conjugated goat anti-mouse secondary antibody (Invitrogen), and Hoechst

33342 (Invitrogen). Images were acquired with a SPOT camera (Diagnostics

Instruments) mounted on an Olympus IX51 microscope (Olympus).

References 1. X. M. Yin et al., Nature 400, 886 (Aug 26, 1999). 2. O. Takeuchi et al., Proc Natl Acad Sci U S A 102, 11272 (Aug 9, 2005). 3. J. R. Jeffers et al., Cancer Cell 4, 321 (Oct, 2003). 4. F. Tronche et al., Nat Genet 23, 99 (Sep, 1999). 5. T. M. Miller, E. M. Johnson, Jr., J Neurosci 16, 7487 (Dec 1, 1996). 6. E. H. Cheng et al., Mol Cell 8, 705 (Sep, 2001). 7. E. H. Cheng, T. V. Sheiko, J. K. Fisher, W. J. Craigen, S. J. Korsmeyer, Science

301, 513 (Jul 25, 2003). 8. H. Kim et al., Nat Cell Biol 8, 1348 (Dec, 2006).

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Fig. S1. The transmembrane domain of PUMA is important for its cytochrome c releasing activity. Isolated mitochondria were incubated with IVTT vector control, PUMA, or PUMA∆C (aa1-164) for 30 min at 30oC, after which the release of cytochrome c was quantified by ELISA assays. The levels of indicated IVTT proteins were analyzed by Nu-PAGE and autoradiography.

Fig. S2. Although Bax-deficient cerebellar granule neurons are more resistant to IR than Bak-deficient neurons, caspases are still activated. Immunohistochemistry for cleaved caspase-3 from cerebella of postnatal day 5 (P5) mice of the indicated genotypes that were irradiated with 14 Gy γ-irradiation. (A) Bak-/-, 18 hours post-IR. (B) Bax-/-, 18 hours post-IR. (C) Bax-/-, 30 hours post-IR. Arrows denote the external granular layer of cerebellum. Data shown are representative images of two to three independent experiments.

Fig. S3. Expression of BCL-2 family proteins in thymocytes (T) or cultured cerebellar granule neurons (CGN). Cell lysates from thymocytes or cultured CGN were assessed by Western blots using the indicated antibodies. A cross-reactive band serves as a loading control.

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Fig. S4. Bid-/-Bim-/-Puma-/- TKO cerebellar granule neurons are completely resistant to potassium-deprivation induced apoptosis. Cerebellar granule neurons from WT, Bim-/-, Puma-/-, Bim-/-Puma-/-, or Bid-/-Bim-/-Puma-/- mice were cultured in high-K+ (K25+S) for 7 days, then transferred to low-K+ medium (K5+S) to induce apoptosis. Photographs of phase contrast images were taken 72 hours after the media were switched. (A) WT, high-K+. (B) WT, low-K+. (C) Bim-/-, high-K+. (D) Bim-/-, low-K+. (E) Puma-/-, high-K+. (F) Puma-/-, low-K+. (G) Bim-/-Puma-/-, high-K+. (H) Bim-/-Puma-/-, low-K+. (I) Bid-/-Bim-/-

Puma-/-, high-K+. (J) Bid-/-Bim-/-Puma-/-, low-K+.

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Fig. S5. Bid-/-Bim-/-Puma-/- TKO cerebellar granule neurons are completely resistant to potassium-deprivation induced apoptosis. Cerebellar granule neurons from WT, Bim-/-, Puma-/-, Bim-/-Puma-/-, or Bid-/-Bim-/-Puma-/- mice were cultured in high-K+ (K25+S) for 7 days, then transferred to low-K+ medium (K5+S) to induce apoptosis. Photographs of propidium iodide (PI) staining images were taken 72 hours after the media were switched. (A) WT, high-K+. (B) WT, low-K+. (C) Bim-/-, high-K+. (D) Bim-/-, low-K+. (E) Puma-/-, high-K+. (F) Puma-/-, low-K+. (G) Bim-/-Puma-/-, high-K+. (H) Bim-/-Puma-/-, low-K+. (I) Bid-/-Bim-/-Puma-/-, high-K+. (J) Bid-/-Bim-/-Puma-/-, low-K+.

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Fig. S6. Triple deficiency of Bid, Bim, and Puma results in enlarged thymi, splenomegaly, and lymphadenopathy. (A) A representative photograph of thymi from sex-matched WT, Bid-/-Bim-/- DKO, or Bid-/-Bim-/-Puma-/- TKO mice at 6-8 weeks of age. DKO and TKO mice were littermates. (B) A representative photograph of spleens from sex-matched WT, Bid-/-Bim-/- DKO, or Bid-/-Bim-/-Puma-/- TKO mice at 6-8 weeks of age. DKO and TKO mice were littermates. (C) A representative photograph of lymph nodes from WT, Bid-/-Bim-/- DKO, or Bid-/-Bim-/-Puma-/- TKO mice at 9-11 weeks of age.

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Fig. S7. Triple deficiency of Bid, Bim, and Puma results in accumulation of thymocytes and splenocytes. (A) Numbers of total thymocytes (mean ± S.D.) from wild-type (n=3), Bid-/-Bim-/- (DKO, n=3), or Bid-/-Bim-/-Puma-/- (TKO, n=3) mice at 6 to 8 weeks of age. (B) Spleen weight (mean ± S.D.) of wild-type (n=4), Bid-/-Bim-/- (DKO, n=3), or Bid-/-Bim-/-

Puma-/- (TKO, n=4) mice at 6 to 9 weeks of age. (C) Numbers of total splenocytes (mean ± S.D.) from wild-type (n=4), Bid-/-Bim-/- (DKO, n=3), or Bid-/-Bim-/-Puma-/- (TKO, n=4) mice at 6 to 9 weeks of age.

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Fig. S8. Triple deficiency of Bid, Bim, and Puma results in accumulation of myeloid and lymphoid cells. The number of while blood cells (WBC, x 106/ml), neutrophils (x 106/ml), lymphocytes (x 106/ml), and red blood cells (RBC, x 109/ml) was determined from wild-type (n=4), Bid-/-Bim-/- (DKO, n=2), or Bid-/-Bim-/-Puma-/- (TKO, n=2) mice at 7 to 9 weeks of age using HEMAVET (Drew Scientific). T or B cells were determined by FACS analyses of CD3 or B220 staining.

Fig. S9. Protein expression in thymocytes from WT or Bid-/-Bim-/-Puma-/- TKO mice. Asterisks indicate cross-reactive proteins serving as loading controls.

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Fig. S10. Puma-deficient thymocytes are less resistant to apoptosis than Bid-/-Bim-/-Puma-/- TKO cells. (A to E) Thymocytes from WT (n=3) or Puma-/- (n=3) mice at 6 to 8 weeks of age were cultured under the following conditions: in the absence of cytokine (A), after exposure to 2.5 Gy γ-irradiation (B), in the presence of etoposide (C), in the presence of tunicamycin (D), or in the presence of dexamethasone (E). Cell death was quantified by annexin-V staining at the indicated times. Data are the mean percentage of annexin-V-positive cells ± SD from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

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Fig. S11. Cleavage of BID upon diverse apoptotic signals. Cell lysates from thymocytes that were untreated, irradiated with γ-irradiation (2.5 Gy), or treated with tunicamycin or agonistic anti-Fas antibody were assessed by anti-BID Western blot. A cross-reactive band served as a loading control.

Fig. S12. Triple deficiency of Bid, Bim, and Puma prevents the translocation of cytochrome c. Fluorescence microscopy of WT or Bid-/-Bim-/-Puma-/- thymocytes 20 hours after exposure to 2.5 Gy γ-irradiation was performed to detect cytochrome c localization. Analyses of multiple fields summarize percent of cells displaying cytochrome c translocation. *, P<0.05.

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Fig. S13. Triple deficiency of Bid, Bim, and Puma prevents the translocation of cytochrome c. Fluorescence microscopy of WT or Bid-/-Bim-/-Puma-/- primary mouse embryonic fibroblasts 36 hours after exposure to tunicamycin. Red represents cytochrome c immunostaining and blue is Hoechst staining.

Fig. S14. Mitochondria isolated from Bid-/-Bim-/-Puma-/- mouse embryonic fibroblasts were incubated with the indicated IVTT BH3-only proteins for 30 min at 30oC, after which the release of cytochrome c was quantified by ELISA assays. Data shown are mean ± SD from three independent experiments. *, P<0.05.

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Fig. S15. Bid-/-Bim-/-Puma-/- TKO cell are resistant to the BAD mimetic, ATB-737. (A) CD19+ B cells purified from the bone marrow of WT or Bid-/-Bim-/-Puma-/- TKO mice were transformed with c-Myc/BCL-2 by retroviral transduction. The transformed B cells were mock treated or treated with 0.1 µM ABT-737 for 24 hours and cell death was quantified by annexin-V staining. (B) Primary mouse embryonic fibroblasts isolated from WT or Bid-/-Bim-/-Puma-/- TKO mice were infected with retrovirus expressing the indicated genes for 18 hours, then treated with 1 µM ABT-737 for 8 hours. Cell death was quantified by annexin-V staining. Data are the mean percentage of annexin-V-positive cells ± SD from three independent experiments. *, P<0.05.

Genotype Total genotyped

Expected Number

Actual Number

% Expected

Bid-/-Bim-/- 62 16 13 81 Bid-/-Bim-/-Puma-/- 372 42 21 50

Table S1. Targeted deletion of Bid, Bim, and Puma causes partial embryonic lethality. To generate Bid-/-Bim-/- mice, Bid+/-Bim+/- mice were crossed to Bid+/-Bim+/- mice, or Bid-/-

Bim+/- mice were crossed to Bid-/-Bim+/- mice. 62 mice were genotyped at 2 weeks after birth. To generate Bid-/-Bim-/-Puma-/- mice, Bid+/-Bim+/-Puma+/- mice were crossed with Bid+/-Bim+/-Puma+/- mice, Bid-/-Bim+/-Puma+/- mice were crossed with Bid-/-Bim+/-Puma+/- mice, Bid+/-Bim+/-Puma-/- mice were crossed with Bid+/-Bim+/-Puma-/- mice, or Bid-/-Bim+/-

Puma-/- mice were crossed with Bid-/-Bim+/-Puma-/- mice. 372 mice were genotyped at 2 weeks after birth.

Genotype Persistent Interdigital Webs Imperforate Vagina Bid-/-Bim-/- 0 % (0/10) 0% (0/5)

Bid-/-Bim-/-Puma-/- 100% (13/13) 67% (4/6) Table S2. Bid, Bim, and Puma triple knockout mice display persistent interdigital webs and imperforate vagina.