Fig. 3.

Gene expression analysis of cells exposed to high IL-7 doses. (A) Panels compare the relative gene expression of IL-7 Tg and WT. Left panel, LRF expression in LSK-Flt3⁺ cells; middle panel, Bcl11b and TCF-7 expression in DN1 c-Kit⁺ cells; right panel, EBF and Pax5 expression in DN1 c-Kit⁺ cells (*P < 0.001). (B) Left panel, relative expression levels of Hes-1 and Deltex expression in DN1 c-Kit⁺ cells from WT and IL-7 Tg mice (*P < 0.0001 for Hes-1 and 0.0008 for Deltex). Right panel, relative expression levels of Hes-1 and Deltex expression in DN1 c-Kit⁺ cells sorted from WT LSK-Flt3⁺ and cultured with OP9-DL1 cells at 1, 10 or 40ng ml⁻¹ of IL-7 (Hes-1 *P < 0.05, Deltex *P < 0.0001). Data shown in (A) n = 8–9 mice each group done in triplicate and (B) are from two experiments done in triplicate. (C) LSK-Flt-3⁺ progenitors were sorted from IL-7 Tg and WT mice. WT cells were cultured on OP9-DL1 stromal cells with 1ng ml⁻¹ (WT-1) or 40ng ml⁻¹ of IL-7 (WT-40). IL-7 Tg cells were cultured on OP9-DL1 stromal cells with 1ng ml⁻¹ IL-7 (IL-7 Tg). After culture, DN1 c-Kit⁺, DN2 c-Kit⁺ and DN3 c-Kit⁻ progenitors were separately sorted then mixed in the same proportions for each group. Total and phospho-Akt Ser⁴⁷³ are shown for all groups. Phospho-Akt-Thr³⁰⁸ is shown in IL-7 Tg and WT-derived cells. (D) Normalized SOCS-1 expression in sorted DN1-c-Kit⁺, DN2-ckit⁺ and DN3-c-Kit⁻ from WT LSK-Flt-3⁺ and OP9-DL1 cell co-cultures treated with1ng ml⁻¹ or 40ng ml⁻¹ of IL-7. IL-7 Tg LSK-Flt3⁺ were cultured with 1ng ml⁻¹ of IL-7 (*P < 0.0001). (E) Normalized SOCS-1 expression in sorted DN3-c-Kit⁻ and DN4-c-Kit⁻ thymocytes from IL-7 Tg and WT mice (*P = 0.0004; **P = 0.0001). (F) STAT5 intracellular staining in DN thymocytes from IL-7 Tg and WT mice. The black filled histograms show the isotype control for STAT5 staining; the grey histograms show STAT5 staining for unstimulated cells; the open histograms correspond to STAT5 staining in progenitors stimulated with IL-7. Gates were DN1-c-Kit⁺, DN2-ckit⁺, DN3-c-Kit⁻ and DN4-c-Kit⁻ within the Lin⁻ population. The experiment was repeated three times.