JAK/STAT and c-kit signaling in Cbfb^rss/− T cells. (A) Expression of IL-7Rα (CD127) and γ_c (IL2RG/CD132) on the DN2 thymocytes from Figure 2C (17.5 dpc). Data are representative of 4 experiments. (B) DN2 cells were sorted from OP9-DL1 cultures (culture conditions and staining as described in Figure 2A). The purity of postsort populations was more than 95%. Expression of individual genes was normalized to Hprt. Values are averaged from triplicate samples from 3 independent experiments. *indicates significant difference (P≤ .05). (C) pStat5 levels in 17.5 dpc DN2 thymocytes on ex vivo stimulation with 0, 1, and 5 ng/mL IL-7 for 20 minutes. Shown is the MFI of the difference between pStat5 under IL-7–stimulated and nonstimulated conditions. The data are averaged from 5 Cbfb^+/+ and 5 Cbfb^rss/− fetuses. Differences are significant at P < .001. (D) Rescue of T-cell development from Cbfb^rss/−Lin⁻ FL cells with CBFβ, but not wild-type or constitutively active Stat5a (S711F; Stat5a^F), or MigR1 alone (−) (n = 5). Cells were harvested after 2 weeks. The expansion of Cbfb^rss/− cells (left graph) is calculated by dividing the number of GFP⁺ cells on day 14 by that on day 7 of culture. Only CBFβ expression significantly (*P≤ .001) increased cell numbers compared with MigR1-transduced Cbfb^rss/− cells. The right-hand graph shows the percentage of GFP⁺ cells expressing CD4 and CD8. (E) Expression of cell surface c-kit on 17.5 dpc DN1 cells. Bar graph on left is the percentage of c-kit⁺ DN1 (Lin⁻ as in Figure 2B; CD45⁺CD44⁺CD25⁻) thymocytes, and on right is the MFI of c-kit staining on c-kit⁺ DN1 thymocytes. The data are averaged from 4 Cbfb^+/+ and 4 Cbfb^rss/− fetuses. (F) Percentage of DN1 (CD44⁺CD25⁻CD45⁺ Lin⁻) cells expressing surface c-kit and MFI of c-kit staining on c-kit⁺ DN1 cells after 6 days of OP9-DL1 culture. The data are averaged from 4 Cbfb^+/+ and 3 Cbfb^rss/− fetuses.