Fig. 3.

Proliferative and apoptotic responses in Mst1-null and wild-type T cells. (A) (³H) thymidine incorporation into Mst1-null and wild-type T cells. Wild-type (solid line) or Mst1-null (dashed line) splenic T cells purified by CD90 (Thy 1.2) microbeads were stimulated with plate-bound anti-CD3 (1 μg/ml) without or with anti-CD28 (1 μg/ml). (³H) thymidine incorporation was determined in triplicate at the times indicated. One of four experiments with similar results is shown. (B) (³H) thymidine incorporation into T cell subsets. Purified CD4⁺ CD62Lhi (naïve) (Left) and CD62Llo cells (Right) were isolated from the spleens of wild-type (black bars) and Mst1-null (white bars) mice. (³H) thymidine incorporation was determined after incubation for 48 h with anti-CD3 or anti-CD3⁺ anti-CD28. The results shown are the mean and standard errors of triplicate determinations from one of four independent experiments with similar results. (C) Mst1 polypeptide and mRNA are greatly reduced in wild-type effector/memory compared with wild-type naïve CD4⁺ cells. The whole cell lysates prepared from naïve and effector/memory CD4⁺ cells were analyzed by immunoblot with anti-Mst1, -Mst2, -Nore1B, and -Mob1. Actin served as a loading control. RNA was isolated from these cells and real-time PCR performed with specific primers to Mst1 and CD25. (D) Annexin V binding to freshly isolated total and naïve T cells from Mst1-null and wild-type littermates. Annexin V+/7AAD-staining of lymphocytes freshly isolated from spleen, peripheral blood, lymph node, and Peyer's patches of 8- to 9-week wild-type (black bars) and Mst1-null (white bars) mice. The bars represent the mean percentage and SD of Annexin V+/7AAD- cells from four independent experiments. (E) Annexin V binding by T cells after stimulation in vitro. Annexin V+/7AAD-staining of naïve and memory CD4⁺ T cells stimulated with anti-CD3 or anti-CD3⁺ anti-CD28 for 48 h. Shown is the mean and SE of triplicate measurements from one of two experiments.