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1 Supplementary Materials for The VDAC2-BAK Rheostat Controls Thymocyte Survival Decheng Ren, Hyungjin Kim, Ho-Chou Tu, Todd D. Westergard, Jill K. Fisher, Jeff A. Rubens, Stanley J. Korsmeyer, James J.-D. Hsieh, Emily H.-Y. Cheng* *To whom correspondence should be addressed. Published 25 August 2009, Sci. Signal. 2, ra48 (2009) DOI: /scisignal This PDF file includes: Fig. S1. VDAC1 and VDAC2 are found in a wide range of mouse tissues. Fig. S2. Abundance of VDAC2 by genotype. Fig. S3. Analysis of thymocyte numbers by genotype. Fig. S4. Effect of the deficiency in Vdac2 on the abundance of other VDAC isoforms and of pro- and antiapoptotic proteins. Fig. S5. Conditional deletion of Vdac2 results in enhanced apoptosis in DP thymocytes. Fig. S6. WT and Vdac2 null thymocytes display comparable kinetics of BIM induction. Fig. S7. Deficiency in Bak rescues the apoptotic phenotype associated with deletion of Vdac2. Fig. S8. Deficiency in Bax fails to rescue the apoptotic phenotype associated with deletion of Vdac2. Fig. S9. Deficiency in both Vdac2 and Bak does not affect homeostasis or survival of thymocytes. Fig. S10. Vdac2 null thymocytes display an enhanced apoptotic response to TCR stimulation, which is rescued by deficiency in Bak, but not Bax. Fig. S11. PCR analysis of Vdac2 deletion in thymocyte subsets.

2 Fig. S1. VDAC1 and VDAC2 are found in a wide range of mouse tissues. Cell lysates (30 μg) from the indicated mouse tissues were analyzed by Western blotting with antibodies against VDAC2 or VDAC1. Data shown are representative of two experiments.

3 Fig. S2. Abundance of VDAC2 by genotype. Cell lysates (30 μg) from mice with the indicated genotypes were analyzed by Western blotting with an antibody against VDAC2.

4 Fig. S3. Analysis of thymocyte numbers by genotype. Numbers of total thymocytes (mean ± SD) from sex-matched mice of the indicated genotypes at 6 to 8 weeks of age (n = 6 for WT mice, n = 6 for Vdac2 f/+ Lck-Cre - mice, and n = 3 for Vdac2 +/+ Lck-Cre + mice).

5 Fig. S4. Effect of the deficiency in Vdac2 on the abundance of other VDAC isoforms and of proand anti-apoptotic proteins. Data shown are representative of two experiments.

6 Fig. S5. Conditional deletion of Vdac2 results in enhanced apoptosis in DP thymocytes. (A) Thymocytes from Vdac2 WT (Vdac2 f/+ Lck-Cre -, n = 2) or Vdac2 KO (Vdac2 f/- Lck-Cre +, n = 2) sex-matched littermate mice at 6 to 8 weeks of age were cultured in the absence of cytokines. (B to D) Thymocytes from the mice indicated in (A) were cultured for the indicated times after exposure to 2.5 Gy of γ-irradiation (B), were treated with etoposide (C), or were treated with dexamethasone (D). In each case, cells were stained with PE-conjugated anti-cd4 antibody, FITC-conjugated anti-cd8 antibody, and APC-conjugated annexin-v at the indicated times and analyzed for the percentage of DP thymocytes that were positive for annexin-v. Data shown are the mean percentage ± SD of annexin V-positive cells *, P<0.05; **, P<0.01.

7 Fig. S6. WT and Vdac2 null thymocytes display comparable kinetics of BIM induction. Cell lysates from Vdac2 WT (Vdac2 f/+ Lck-Cre - ) or Vdac2 KO (Vdac2 f/- Lck-Cre + ) thymocytes treated with ionomycin for the indicated times were analyzed by Western blotting with antibodies specific for BIM or actin. The blot incubated with the antibody against cleaved caspase-3 that was shown in Fig. 2D was stripped and incubated with the anti-bim antibody.

8 Fig. S7. Deficiency in Bak rescues the apoptotic phenotype associated with deletion of Vdac2. (A) Numbers of DN, CD4 SP, CD8 SP, or DP T cells from the thymi of Vdac2 KO (Vdac2 f/- Bak +/+ Lck-Cre +, n = 8) or Vdac2, Bak DKO (Vdac2 f/- Bak -/- Lck-Cre +, n = 5) sex-matched littermate mice at 6 to 8 weeks of age. Data presented are the mean ± SD. (B to E) Vdac2 KO or Vdac2, Bak DKO thymocytes were cultured in the absence of cytokines (B), were cultured for the indicated times after exposure to 2.5 Gy of γ-irradiation (C), were treated with etoposide (D), or were treated with dexamethasone (E) and cell death was quantified by staining with annexin- V at the indicated times. Data are the mean percentage ± SD of cells that were positive for annexin-v from 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

9 Fig. S8. Deficiency in Bax fails to rescue the apoptotic phenotype associated with deletion of Vdac2. (A) Numbers of DN, CD4 SP, CD8 SP, or DP T cells from the thymi of Vdac2 KO (Vdac2 f/- Bax +/+ Lck-Cre +, n = 7) or Vdac2, Bax DKO (Vdac2 f/- Bax f/- Lck-Cre +, n = 7) sex-matched littermate mice at 6 to 8 weeks of age. Data presented are the mean ± SD. (B to E) Vdac2 KO or Vdac2, Bax DKO thymocytes were cultured in the absence of cytokines (B), were cultured for the indicated times after exposure to 2.5 Gy of γ-irradiation (C), were treated with etoposide (D), or were treated with dexamethasone (E) and cell death was quantified by staining with annexin- V at the indicated times. Data shown are the mean percentage ± SD of cells that were positive for annexin-v from 3 independent experiments. (F) Analysis of a Western blot of thymocyte lysates from mice of the indicated genotypes with an anti-bax antibody. The same membrane was stripped and incubated with an anti-actin antibody.

10 Fig. S9. Deficiency in both Vdac2 and Bak does not affect homeostasis or survival of thymocytes. (A) Numbers of total thymocytes, DN, CD4 SP, CD8 SP, or DP T cells from thymi of Bak KO (Vdac2 f/+ Bak -/- Lck-Cre -, n = 5) or Vdac2, Bak DKO (Vdac2 f/- Bak -/- Lck-Cre +, n = 4) sex-matched littermate mice at 6 to 8 weeks of age. Data presented are the mean ± SD. (B) A representative photograph of thymi from sex-matched littermate mice of the indicated genotypes. (C to F) Thymocytes from mice indicated in (A) were cultured in the absence of cytokines (C), were cultured for the indicated times after exposure to 2.5 Gy of γ-irradiation (D), were treated with etoposide (E), or were treated with dexamethasone (F) and cell death was quantified by annexin-v staining at the indicated times. Data shown are mean percentage ± SD of annexin-vpositive cells from 3 independent experiments.

11 Fig. S10. Vdac2 null thymocytes display an enhanced apoptotic response to TCR stimulation, which is rescued by deficiency in Bak, but not Bax. Vdac2 WT (Vdac2 f/+ Lck-Cre - ), Vdac2 KO (Vdac2 f/- Lck-Cre + ), Vdac2, Bak DKO (Vdac2 f/- Bak -/- Lck-Cre + ), Vdac2, Bax DKO (Vdac2 f/- Bax f/- Lck-Cre + ), or Bak KO (Bak -/- ) mice at 6 to 10 weeks of age were injected intraperitoneally with anti-cd3ε antibody. Numbers of (A) total thymocytes, (B) DN, (C) CD4 SP, and (D) CD8 SP thymocytes from mice of the indicated genotypes 48 hours after intraperitoneal injection with anti-cd3ε antibody are shown. Data shown are the mean ± SD from 3 independent experiments.

12 Fig. S11. PCR analysis of Vdac2 deletion in thymocyte subsets. Thymocytes from Vdac2 WT (Vdac2 f/+ Lck-Cre - ) or Vdac2 KO (Vdac2 f/- Lck-Cre + ) littermate mice were stained with FITCconjugated anti-cd8 antibody and PE-conjugated anti-cd4 antibody and then subjected to highspeed cell sorting. Genomic DNA from DN, DP, CD4 SP, or CD8 SP thymocytes was analyzed by PCR to determine the representation of Vdac2 alleles as well as of Lck-Cre.