Fig. 6.

GSK3 inhibition enhances survival of CD4+CD8+ thymocytes in response to negative selecting TCR signals. (A) Purified DP thymocytes were cultured in the presence or absence of plate-bound anti-TCR/CD2/CD28 antibodies and the presence or absence of 60 mM lithium for 4 h. Cells were fixed, permeabilized, stained for cleaved caspase 3 and analyzed by flow cytometry. Results from the four conditions are shown as an overlay histogram. TCR-stimulated DP thymocytes were the only group that showed an increase in the frequency of cleaved caspase 3 ± cells. All three other groups (unstimulated DP in the presence or absence of lithium and stimulated DP in the presence of lithium) showed essentially the same frequency of cleaved caspase 3+ cells (∼11%). These results are representative of at least three experiments. (B) In a different experiment, purified DP and SP thymocytes were subject to the same treatment as in (a), lysed and analyzed for cleaved caspase 3 by western blot. Relative intensities of bands (cleaved caspase 3/Akt) are indicated.