Figure 6

Itk ^-/- thymocytes are more sensitive than wild type thymocytes to apoptosis induced by dexamethasone but not by TCR activation or heat shock. (A) Itk ^-/- (KO) and wild type (WT) thymocytes were left untreated or treated with 10^-7 M dexamethasone (DEX) for 18 h before staining with a hypotonic solution of propidium iodide followed by flow cytometric analysis for apoptotic cells. One representative experiment performed on cells pooled from four mice is shown. The percentage of apoptotic cells within the gate is reported. (B) The percentage of apoptotic cells following treatment with the specified concentration of DEX is represented as the mean (±SD) of four independent experiments, performed on cells pooled from three to four mice. Because the level of spontaneous apoptosis was higher in KO than WT thymocytes, apoptosis in DEX-treated cells was normalized using the respective level of spontaneous apoptosis and the DEX-specific apoptosis was calculated as specified in Methods (right panel). (C) KO and WT thymocytes were treated with anti-CD3 antibody for 18 h and apoptosis was evaluated as above. The percentage of apoptotic cells is represented as the mean (±SD) of four independent experiments, with each experiment performed on cells pooled from three or four mice. Specific apoptosis is shown in the right panel. (D) KO and WT thymocytes were incubated at 43°C for the specified times and apoptosis was evaluated after a further 18 h as described above. The percentage of apoptotic cells is represented as the mean (±SD) of four independent experiments, with each experiment performed on cells pooled from three or four mice. Specific apoptosis is shown in the right panel. *P < 0.05, **P < 0.01, and ***P < 0.001, according to Student’s t-test comparing KO with WT apoptosis (left panel) or DEX-specific apoptosis (right panel).