Fig. 5: Activity of caspase-3, but not caspase-8 or caspase-9, is regulated by the glycolytic state of effector T cells

Activated T cells were cultured for 3 days in IL-2 with or without 5 mM 2-DG. a Active caspases were selectively precipitated using biotin-VAD (bVAD) and streptavidin Sepharose beads, and immunoblotted for caspases-8, -9, -7, and -3 (the blot is representative of two independent experiments). b Densitometry of active caspase-8, -9, -7, and -3 in (a; for caspase-8, -9, and -3: two-way ANOVA with Sidak’s correction; for caspase-7: unpaired t-test; NS not significant; *p < 0.05; mean ± S.D. of two replicates). c Cell death was determined by Live/Dead staining after centrifugation over Histopaque (unpaired t-test; mean ± S.D.; **p < 0.01; n = 3 independent experiments). d Restimulation-induced cell death was promoted by 16 h anti-CD3 restimulation of IL-2-cultured T cells cultured with or without 2-DG, and cell death determined by Live/Dead staining using flow cytometry. Data indicate fold change compared to the unstimulated medium control (two-way ANOVA with Sidak’s correction; NS not significant; *p < 0.05; mean ± S.D. of two replicates; n = 2 independent experiments). e Cell lysates of T cells grown in the indicated conditions were immunoblotted for FasL. Controls included FasL-transfected 3T3 cells or mock-transfected 3T3 cells (the immunoblot is representative of two independent experiments). f Densitometry of FasL in (e; one-way ANOVA; *p < 0.05; **p < 0.01; mean ± S.D. of two replicates)