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: [email protected]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
15
Embed
Supporting Online Material for · 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
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
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).
5
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.
6
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-/-
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+.
8
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.
9
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.
10
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.
11
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.
12
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.
13
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.
14
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.
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.