PMC

FACS (fluorescence-activated cell sorting) analyses of thymocytes after stimulation of the TCR in vivo. Total thymocytes were isolated from 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 (6 to 10 weeks of age) 48 hours after intraperitoneal injection with anti-CD3ε or PBS (control). Cells were stained with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8 and analyzed by flow cytometry. The percentages of the different thymocyte subpopulations in gated live cells are shown within the quadrants. Data shown are representative of three independent experiments.

Strategy for conditional knockout of Vdac2. (A) Schematics of the murine Vdac2 locus (top) and the targeting construct (bottom). The targeted allele was first derived in RW4 ES cells by homologous recombination, followed by transient expression of Cre recombinase to generate the flox or null alleles. Red triangles, LoxP sites. (B) Schematics of Vdac2 alleles. Red triangles, LoxP sites; black arrowheads, PCR primers. Primers A and C amplify only the null allele. (C) Genomic DNA from thymocytes or liver was analyzed by PCR to determine the representation of Vdac2 alleles as well as that of Lck-Cre. (D) Analysis of a Western blot of thymocyte lysates from mice of the indicated genotypes with an antibody against VDAC2. A cross-reactive protein served as a loading control.

Deficiency in Bak but not Bax rescues the apoptotic phenotype associated with deletion of Vdac2. (A) Number of total thymocytes (represented as the mean ± SD) from sex-matched littermate mice of the indicated genotypes at 6 to 8 weeks of age (n = 8 for Vdac2^f/–Bak^+/+Lck-Cre⁺ mice and n = 5 for Vdac2^f/–Bak^–/–Lck-Cre⁺ mice). (B) A representative photograph of thymi from sex-matched littermate mice of the indicated genotypes. (C) Numbers of total thymocytes (represented as the mean ± SD) from sex-matched littermate mice of the indicated genotypes at 6 to 8 weeks of age (n = 8 for Vdac2^f/–Bax^+/+Lck-Cre⁺ mice and n = 9 for Vdac2^f/–Bax^f/–Lck-Cre⁺ mice). (D) A representative photograph of thymi from sex-matched littermate mice of the indicated genotypes. (E) Vdac2 KO or Vdac2, Bak DKO thymocytes were treated with ionomycin and cell death was quantified by staining with annexin V at the indicated times. Data shown are the mean percentage of annexin V–positive cells ± SD from n = 3 experiments. (F) Vdac2 KO or Vdac2, Bax DKO thymocytes were treated with ionomycin and cell death was quantified by staining with annexin V at the indicated times. Data shown are the mean percentage of annexin V–positive cells ± SD from n = 3 experiments. (G) Number of total thymocytes (mean ± SD) from sex-matched littermate mice of the indicated genotypes at 6 to 8 weeks of age (n = 5 for Vdac2^f/+Bak^–/–Lck-Cre^– mice and n = 4 for Vdac2^f/–Bak^–/–Lck-Cre⁺ mice). (H) Bak KO or Vdac2, Bak DKO thymocytes were treated with ionomycin and cell death was quantified by staining with annexin V at the indicated times. Data shown are the mean ± SD. from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Vdac2 null thymocytes display early homo-oligomerization of BAK, translocation of cytochrome c, and activation of caspases upon apoptosis. (A) Thymocytes from Vdac2 WT (Vdac2^f/+Lck-Cre^–) or Vdac2 KO (Vdac2^f/–Lck-Cre⁺) littermate mice were left untreated or were treated with ionomycin for 6 hours. Protein lysates were subjected to Superdex 200 (HR10/30) gel-filtration chromatography. Fractions were analyzed by Western blotting with an antibody against BAK. Data shown are representative of two experiments. (B) Fluorescence microscopy of Vdac2 WT (Vdac2^f/+Lck-Cre^–) or Vdac2 KO (Vdac2^f/–Lck-Cre⁺) thymocytes treated with ionomycin for 6 hours. Red represents cytochrome c immunostaining and blue is Hoechst staining. Arrowheads denote cells displayingdiffuse cytosolicstaining for cytochrome c or loss of cytochrome c staining, which is indicative of translocation of cytochrome c. Images shown are representative of three independent experiments. (C) Analyses of multiple fields from experiments shown in (B) summarize the percentage of cells displaying translocation of cytochrome c. **P < 0.05. (D) 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 assessed by Western blotting with antibodies specific for PARP, cleaved PARP, cleaved caspase-3, or actin. Data shown are representative of two experiments.

Vdac2 null thymocytes display an enhanced apoptotic response to stimulation of the TCR, which is rescued by a deficiency in Bak but not Bax. (A) Thymocytes from Vdac2 WT (Vdac2^f/+Lck-Cre^–), Vdac2 KO (Vdac2^f/–Lck-Cre⁺) or Vdac2, Bak DKO (Vdac2^f/–Bak^–/–Lck-Cre⁺) littermate mice at 6 to 10 weeks of age were cultured in plates coated with anti-CD3ε. Cell death was quantified by staining with annexin V at the indicated times. Data shown are the mean ± SD from three independent experiments. (B) Vdac2 null thymocytes display early homo-oligomerization of BAK in response to stimulation of the TCR. Thymocytes from Vdac2 WT (Vdac2^f/+Lck-Cre^–) or Vdac2 KO (Vdac2^f/–Lck-Cre⁺) littermate mice were cultured in plates coated with anti-CD3ε for 14 hours. Protein lysates were subjected to Superdex 200 (HR10/30) gel-filtration chromatography. Fractions were analyzed by Western blotting with an antibody against BAK. (C) A representative photograph of thymi from sex-matched littermate mice of the indicated genotypes at 48 hours after intraperitoneal injection of anti-CD3e antibody or PBS as a negative control. (D) A representative photograph of thymi from sex-matched littermate mice of the indicated genotypes at 48 hours after intraperitoneal injection of anti-CD3ε or PBS (negative control). (E) Numbers of DP thymocytes that remain viable from 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 (6 to 10 weeks of age) 48 hours after intraperitoneal injection with anti-CD3ε. Data shown are the mean ± SD from three independent experiments.

Conditional deletion of Vdac2 results in loss of thymocytes, which is associated with enhanced apoptosis. (A) Numbers of total thymocytes (mean ± SD) from sex-matched littermate mice of the indicated genotypes at 6 to 8 weeks of age. n = 19 for Vdac2^f/+Lck-Cre^– mice, n = 5 for Vdac2^f/+Lck-Cre⁺ mice, n = 5 for Vdac2^f/–Lck-Cre^– mice, and n = 23 for Vdac2^f/–Lck-Cre⁺ mice). (B) Numbers of total thymocytes, DN, CD4 SP, CD8 SP, and DP T cells from the thymi of Vdac2 WT (Vdac2^f/+Lck-Cre^–, n = 7) or Vdac2 KO (Vdac2^f/–Lck-Cre⁺, n = 11) sex-matched littermate miceat 6 to 8 weeks of age. Data presented are the mean thymocyte number ± SD. (C to G) Thymocytes from the mice indicated in (B) were cultured under the following conditions: in the absence of cytokine (C), for the indicated time after exposure to 2.5 Gy of γ-radiation (D), in the presence of etoposide (E), in the presence of dexamethasone (F), or in the presence of ionomycin (G), and in each case, 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.