Figure 1. STIM-dependent store-operated Ca²⁺ entry is dispensable for thymic selection of T cells.

(A) Conventional T cell development in mice transplanted with fetal liver cells isolated from Stim1^−/− (1KO), Stim1^−/− Stim2^−/− (DKO) and wild-type (WT, Stim1^+/+Stim2^+/+) littermate control embryos at embryonic day 14.5. (B) Protein expression of STIM1 (left) and STIM2 (right) in DP thymocytes from (A). (C) Ca²⁺ influx in response to 1 μM thapsigargin (TG) (top, n=5) or anti-CD3 crosslinking (bottom, n=3) in littermate wild-type control (black) and Stim1^−/− Stim2^−/− (grey) DP thymocytes from (A). (D) Flow cytometric analysis of negative selection in male Stim1^fl/flStim2^fl/fl Lck-Cre Rag2^−/− HY TCR-transgenic (tg) mice (top) and frequency of CD8^high T cells in the thymus or lymph nodes (bottom). Control, Stim1^+/+Stim2^+/+ Lck-Cre Rag2^−/− HY TCR; DKO, Stim1^fl/flStim2^fl/fl Lck-Cre Rag2^−/− HY TCR. 3 of six weeks old mice were analyzed in each case. (E) SEB-induced negative selection in Stim1^fl/flStim2^fl/fl Vaν-iCre mice. Frequencies of SEB-sensitive CD24^hiCD4⁺TCRVβ8⁺ and SEB-insensitive CD24^hiCD4⁺TCRVβ6⁺ immature thymocytes were analyzed 2 days after the administration of 200 μg SEB. Control, Stim1^fl/flStim2^fl/fl; DKO, Stim1^fl/flStim2^fl/fl Vaν-iCre. 4 mice were analyzed in each case. (F) Flow cytometric analysis of positive selection in female Stim1^fl/flStim2^fl/fl Lck-Cre Rag2^−/− HY TCR-transgenic (tg) mice (left and center) and frequency of CD8^high T cells in the thymus or lymph nodes (right). Control, Stim1^+/+Stim2^+/+ Lck-Cre Rag2^−/− HY TCR; DKO, Stim1^fl/flStim2^fl/flLck-Cre Rag2^−/− HY TCR. 3 mice were analyzed in each case. Data are representative of two (A, B, C and E) and more than three (D and F) independent experiments. Error bars (C, D, E and F) denote mean ± SEM. *, P<0.001; **, P<0.03; ***, P<0.05; NS, not significant. See also Figure S1.