FIGURE 4.

Apoptosis and cellular organelles in Vps34-deficient T cells. (A,B) Splenic (A), lymph node (A), liver (A) or thymic (B) mononuclear cells were prepared from Vps34^f/f;CD4-Cre or Vps34^f/+;CD4-Cre mice and were stained with anti-CD4 and -CD8 antibodies, 7-AAD and Annexin V. Annexin V⁺7-AAD⁻ cells were considered early apoptotic cells and annexin V⁺7-AAD⁺ cells were considered late apoptotic cells. A representative of 4 independent experiments is shown. The average ± SEM of 6 mice is shown. A summary of these results is shown in Supplemental Fig. 2B. (C) Splenocytes or thymocytes were stained with MitoTracker^® Red (50 nM) or ER-Tracker^™ red (1 μM) at 37°C for 30 min in complete medium followed by surface staining with anti-CD4 and -CD8 antibodies at 4°C. The data shown are gated on splenic CD4⁺ or CD8⁺ T cells or thymic CD4 SP, CD8 SP or CD4 CD8 DP cells. A representative of 3 independent experiments is shown. (D) Absolute number of mitochondria and the total size of the mitochondria were determined in activated T cells using transmission electron microscopy. The Image J software was used to manually mark single mitochondria, measuring their surface area, and normalization against the scale bar from transmission electron microscopy images. The data presented are the total size of all the mitochondria or nucleus (as negative control) normalized against the size of the cell. A representative from 30–35 individually imaged cells in each group is shown to the bottom. Scale bar = 500 nm. *p<0.05. (E) Splenocytes derived from Vps34^f/f;CD4-Cre or Vps34^f/+;CD4-Cre mice were stained with anti-CD4 and -CD8 antibodies followed by intracellular staining with anti-Bcl-2 or -Bcl-xL antibodies or their respective isotype controls. The data shown are gated on CD4⁺ or CD8⁺ T cells. A representative experiment of 3 individual experiments is shown to the left and the average ± SEM of 4 mice is summarized. (F) T cells were activated as described in the legend to Fig. 1 and subjected to immunoblot analysis with anti-Bim, -Bad or -Bax antibodies. (G) The rate of glycolysis was measured in activated T cells by incubating 10⁶ cells with ten μCi of 5-[³H] glucose for 1 hr in RPMI medium. The rate of β-oxidation was measured by culture of 4×10⁶ activated T cells in 0.4 ml of RPMI containing 2 μCi 9,10-[³H] palmitate (MP), 2% of BSA (fatty acid free) and 0.25 mM L-carnitine for 4 hrs.