Fig. 3.
Fig. 3. Increase in apoptosis sensitivity of CEM cells by inhibition of NFκB activation. (A) CEM cells were incubated with TRAIL in the concentrations indicated in the presence (•) or absence (○) of LLnL (2.5 μmol/L, 1 hour of preincubation) for 24 hours. Apoptosis was measured using forward side scatter analysis in FACScan. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in five independent experiments. (B) Jurkat cells were treated as in (A) using LLnL at 6.25 μmol/L. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (C) BJAB cells, either mock-transfected (○, •) or overexpressing a dominant negative FADD mutant protein (□, ▪) were treated as in (A) using LLnL at 25 μmol/L. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (D) CEM cells (3 × 107) were transfected with 20 μg of empty vector pPRB or pPRB containing the cDNA for mutant IκBα (serines on position 32 and 36 were replaced by alanines) and cotransfected with 2 μg of pEGFP. After 48 hours, living cells were separated by Ficoll gradient and stimulated with different concentrations of TRAIL for another 24 hours. Apoptosis was measured by forward side scatter analysis in FACScan cytometer gating on GFP-positive cells. Specific apoptosis was calculated using as control unstimulated, GFP-positive cells transfected with mock or IκBα, respectively. Data are the mean of triplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (E) CEM cells were transfected as in (D) and subsequentially stimulated with TRAIL (0.03 μg/mL), anti–APO-1 (0.3 μg/mL), or TNFα (0.3 μg/mL) for another 24 hours. Specific apoptosis was calculated as in (D). Data are the mean of triplicates with a standard deviation less than 10%. Similar results were obtained in three independent experiments. Similar results were obtained using Jurkat cells.

Increase in apoptosis sensitivity of CEM cells by inhibition of NFκB activation. (A) CEM cells were incubated with TRAIL in the concentrations indicated in the presence (•) or absence (○) of LLnL (2.5 μmol/L, 1 hour of preincubation) for 24 hours. Apoptosis was measured using forward side scatter analysis in FACScan. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in five independent experiments. (B) Jurkat cells were treated as in (A) using LLnL at 6.25 μmol/L. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (C) BJAB cells, either mock-transfected (○, •) or overexpressing a dominant negative FADD mutant protein (□, ▪) were treated as in (A) using LLnL at 25 μmol/L. Data are the mean of duplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (D) CEM cells (3 × 107) were transfected with 20 μg of empty vector pPRB or pPRB containing the cDNA for mutant IκBα (serines on position 32 and 36 were replaced by alanines) and cotransfected with 2 μg of pEGFP. After 48 hours, living cells were separated by Ficoll gradient and stimulated with different concentrations of TRAIL for another 24 hours. Apoptosis was measured by forward side scatter analysis in FACScan cytometer gating on GFP-positive cells. Specific apoptosis was calculated using as control unstimulated, GFP-positive cells transfected with mock or IκBα, respectively. Data are the mean of triplicates with a standard deviation less than 10%. Similar results were obtained in two independent experiments. (E) CEM cells were transfected as in (D) and subsequentially stimulated with TRAIL (0.03 μg/mL), anti–APO-1 (0.3 μg/mL), or TNFα (0.3 μg/mL) for another 24 hours. Specific apoptosis was calculated as in (D). Data are the mean of triplicates with a standard deviation less than 10%. Similar results were obtained in three independent experiments. Similar results were obtained using Jurkat cells.

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