c-Myc expression in response to pre-TCR stimulation. (A) Developmental progression to the DP stage after pre-TCR induction in TetOβ-LTH-Rag1^-/- thymocytes. FACS analysis of thymocytes from neonatal thymic organ cultures derived from mice that had been treated with tetracycline during gestation. Thymic lobes were kept in organ cultures in the absence of tetracycline for the indicated times. Top dot plots show CD4 versus CD8 and bottom dot plots show CD44 versus CD25 of gated lin^- cells. Histograms show intracellular (ic) TCRβ expression in the DP and the DN4 subsets. (B) c-Myc Western blot of thymocytes from LTH-Rag1^-/- and TetOβ-LTH-Rag1^-/- mice that had been treated with tetracycline prior to coculturing with OP9-DL1 cells in tetracycline-free growth medium for 24 hours. Thy1⁺ cells were sorted from the cocultures and total cell lysates were used for Western blotting. Lane 1 shows LTH-Rag1^-/-; lane 2, TetOβ-LTH-Rag1^-/-. (C) FACS analysis of α-CD3 mAb-induced thymocyte development in Rag^-/- mice. Dot plots show thymocyte expression profiles for CD4 versus CD8 (top) and CD44 versus CD25 (bottom, data gated on lin^- cells) from Rag^-/- mice 0, 2, and 4 days after intraperitoneal injection of α-CD3 antibody. (D) c-Myc Western blot from Rag^-/- mice that were injected with 50 μg of α-CD3 mAb at the indicated time points prior to killing. Total thymic lysates were obtained for Western blotting. Results are representative of 4 independent experiments.