Figure 2. TNF induces thymocyte cell death in the absence of IKK activity in vitro. (A) Thymocytes from Ikk1fx/fx Ikk2fx/fx R26REYFP CD4Cre (IKKΔTCD4) mice were cultured overnight with the indicated Tnfrsf member, and viability of the indicated SP subset assessed the next day by FACS. (B) Thymocytes from IKKΔTCD4, Cre -ve littermates (Cre -ve), and Tnfrsf1a−/− donors were cultured overnight with and without TNF (30 ng/ml). Bar chart shows viability among the indicated subsets cultured with addition of PBS (open bars) or TNF (filled bars) as assessed by FACS. (C) Thymocytes from WT and Ikk1fx/fx R26REYFP huCD2iCre donors were cultured overnight in the presence of TNF or IKK2 inhibitor Bl605906, or both. Bar chart shows percentage of viability among the indicated subsets as assessed by FACS. (D) Ikk1fx/fx R26REYFP huCD2iCre thymocytes were cultured for 4 h in the presence of TNF and/or IKK2 inhibitor Bl605906. Density plots are of live dead versus active caspase 8 stain by CD8 SP thymocytes. (E) Thymocytes from Ikk1fx/fx R26REYFP huCD2iCre donors were cultured with IKK2 inhibitor and a titration of TNF. Line graph shows the percentage of maximum TNF-induced death for different concentrations of TNF, above background, for the indicated subsets. Concentrations in figure legend indicate LD50 of TNF for the corresponding subset. (F) Thymocytes from WT or Ikk1fx/fx R26REYFP huCD2iCre donors were pre-incubated with IKK2 inhibitor Bl605906 or DMSO vehicle for 1 h where indicated, and then stimulated with TNF and nuclear extracts prepared at different times. Bar charts represent RelA binding to NF-κB binding oligonuclotides as detected by ELISA and displayed as OD. Dotted line indicates the background signal from blank wells. (G) Thymocytes from pLck-IκB-PEST donors or transgene -ve littermates were cultured in different concentrations of TNF overnight. Line graphs are of percentage dead cells in the indicated subsets from pLck-IκB-PEST donors (IκB-PEST, filled circles) or transgene negative litter controls (WT, open circles). Error bars indicate SD of technical duplicates. Data are representative of two (F), three (G), or more (A–E) independent experiments. *, P < 0.05; ***, P < 0.001.
Figure 2.

TNF induces thymocyte cell death in the absence of IKK activity in vitro. (A) Thymocytes from Ikk1fx/fx Ikk2fx/fx R26REYFP CD4Cre (IKKΔTCD4) mice were cultured overnight with the indicated Tnfrsf member, and viability of the indicated SP subset assessed the next day by FACS. (B) Thymocytes from IKKΔTCD4, Cre -ve littermates (Cre -ve), and Tnfrsf1a−/− donors were cultured overnight with and without TNF (30 ng/ml). Bar chart shows viability among the indicated subsets cultured with addition of PBS (open bars) or TNF (filled bars) as assessed by FACS. (C) Thymocytes from WT and Ikk1fx/fx R26REYFP huCD2iCre donors were cultured overnight in the presence of TNF or IKK2 inhibitor Bl605906, or both. Bar chart shows percentage of viability among the indicated subsets as assessed by FACS. (D) Ikk1fx/fx R26REYFP huCD2iCre thymocytes were cultured for 4 h in the presence of TNF and/or IKK2 inhibitor Bl605906. Density plots are of live dead versus active caspase 8 stain by CD8 SP thymocytes. (E) Thymocytes from Ikk1fx/fx R26REYFP huCD2iCre donors were cultured with IKK2 inhibitor and a titration of TNF. Line graph shows the percentage of maximum TNF-induced death for different concentrations of TNF, above background, for the indicated subsets. Concentrations in figure legend indicate LD50 of TNF for the corresponding subset. (F) Thymocytes from WT or Ikk1fx/fx R26REYFP huCD2iCre donors were pre-incubated with IKK2 inhibitor Bl605906 or DMSO vehicle for 1 h where indicated, and then stimulated with TNF and nuclear extracts prepared at different times. Bar charts represent RelA binding to NF-κB binding oligonuclotides as detected by ELISA and displayed as OD. Dotted line indicates the background signal from blank wells. (G) Thymocytes from pLck-IκB-PEST donors or transgene -ve littermates were cultured in different concentrations of TNF overnight. Line graphs are of percentage dead cells in the indicated subsets from pLck-IκB-PEST donors (IκB-PEST, filled circles) or transgene negative litter controls (WT, open circles). Error bars indicate SD of technical duplicates. Data are representative of two (F), three (G), or more (A–E) independent experiments. *, P < 0.05; ***, P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal