Bcl-2/BH3 exposure was observed in anti-CD3/CD28 and PMA/ionomycin-treated thymocytes. (A) Bcl-2/BH3 and Bcl-2 expression of DP cells from thymocytes cultured for 2, 4, and 6 h with 1.25 ng/ml PMA/0.5 μM ionomycin. Solid lines represent untreated thymocytes, and dotted lines represent treated thymocytes. (B) Bcl-2/BH3 exposure of DP cells from thymocytes cultured for 2, 4, and 6 h with 10 μg/ml anti-CD3/2 μg/ml anti-CD28 (plate-bound) and Bcl-2/BH3 exposure of DN and SP cells treated for 6 h. Here, the shaded area represents isotype control, dotted lines represent treated thymocytes, and solid lines represent treated thymocytes in the presence of leptomycin B. (C) Bcl-2/BH3 exposure of DP cells from Nur77^−/− and Bcl-2^−/− thymocytes cultured for 4 h with 2.5 ng/ml PMA/0.5 μM ionomycin. Shaded area represents isotype control, solid lines represent untreated thymocytes, and dotted lines represent treated thymocytes. Bcl-2/BH3 expression of CD69^high and CD69^low DP cells treated with 2.5 ng/ml PMA/0.5 μM ionomycin for 4 h is also shown. The shaded area represents isotype control, the solid line represents CD69^low DP cells, and the thick line represents CD69^high DP cells. (D) Pretreatment with 20 nM leptomycin B or 25 μM of the ERK5 inhibitor U0126 prevented exposure of the Bcl-2/BH3 domain in DP thymocytes stimulated for 4 h with PMA/ionomycin. Here, the shaded areas represent isotype control, dotted lines represent treated thymocytes, and solid lines represent PMA/ionomycin-treated thymocytes in the presence of leptomycin B or U0126. (E) PMA/ionomycin (PMA/iono) -stimulated thymocytes from lck-Bcl-2 mice were immunoprecipitated with anti-BH3 Bcl-2 antibodies or rabbit IgG, followed by blotting with antibodies specific for Nur77, Bcl-2, or Nor-1. Leptomycin (LMB) was added where indicated (+).

Flow cytometric analysis of CD4, CD8, Bcl-2/BH3, and Bcl-2 staining of thymocytes from HY TCR transgenic mice. (A) Bcl-2/BH3 and Bcl-2 expression of DP cells from female and male HY TCR transgenic mice. The shaded area represents isotype control, solid lines represent female DP cells, and the dotted lines represent male DP cells. (B) Male HY TCR mice show no exposure of the BH3 domain of Bcl-2 in DN or SP cells. Five independent experiments have been done with similar results. All staining of HY TCR transgenic mice is on HY TCR⁺ thymocytes.

Nur77 and Nor-1 localize to the mitochondria and associate with Bcl-2 in anti-CD3/CD28 or PMA/ionomycin-treated thymocytes. (A) Nuclear and mitochondrial fractions of wild-type thymocytes cultured with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 (plate-bound) were isolated and blotted with anti-Nur77 and anti–Nor-1 antibodies. For fraction purity, the blot was also probed with antibodies for lamin B, HSP60, Bcl-2, and cytochrome c. (B) Transcriptional activity of Nur77 and Nor-1 was determined using a luciferase assay (as described in Materials and methods) on 293T cells transiently transfected with the indicated expression plasmids along with a Nur77/Nor-1–dependent luciferase reporter construct and renilla internal control construct. (C) The extent of apoptosis in wild-type thymocytes cultured with 10 μg/ml anti-CD3/2 μg/ml CD28 (plate-bound), 1.25 ng/ml PMA/0.5 μM ionomycin, or 0.5 μM dexamethasone (Dex) was assessed by annexin V staining. Apoptosis was normalized to the untreated controls. (D) Cell lysates from lck-Bcl-2 thymocytes cultured with 2.5 ng/ml PMA/0.5 μM ionomycin were immunoprecipitated (IP) with anti–rabbit IgG (IP crtl), anti-Nur77, and anti–Nor-1 antibodies, followed by blotting with an anti–Bcl-2 antibody. Results shown in A and D represent three to six independent experiments. Data in B and C represent the mean ± SD of three independent experiments. Thymocytes were pretreated for 2 h with 20 nM leptomycin B (LMB) where indicated.

Flow cytometric analysis of CD4, CD8, Bcl-2/BH3, and Bcl-2 staining of thymocytes from F5 TCR transgenic mice. (A) Bcl-2/BH3 and Bcl-2 expression of DP cells from F5 TCR transgenic mice 15 h after injection of PBS or 20 or 50 nmol of nucleocapsid peptide. Here, the shaded area represents isotype control, solid lines represent DP cells from PBS-injected mice, thick solid lines represent DP cells from mice injected with 20 nmol NP peptide, and dotted lines represent DP cells from mice injected with 50 nmol NP peptide. (B) Bcl-2/BH3 exposure was not observed in DN and SP cells from F5 TCR transgenic mice after injection of PBS or 20 or 50 nmol of nucleocapsid peptide. (C) Bcl-2/BH3 expression on CD69^high and CD69^low DP cells from a F5 TCR transgenic animal 10 h after injection with 50 nmol NP peptide. Here, the shaded area represents isotype control, the solid line represents CD69^low DP cells, and the thick solid line represents CD69^high DP cells. Five independent experiments have been done with similar results. All staining of F5 TCR transgenic mice is on Vβ11⁺ thymocytes.