LN DN cells show distinct gene expression profiles. Real-time RT-PCR assays and FACS analysis measuring gene and protein expression in DN1 (A-B), pre-DN2 (C-D), and DN4 (E-F) cells sorted from lymphoid organs. The mRNA levels of the wt thymus for DN1 (A) and DN4 (E) cells and of wt LN for pre-DN2 (C) cells were set as 1. Hprt mRNA levels were used to normalize cDNA content among subpopulations. (A,C,E) Data are mean ± SD from 3 independent experiments. Differences between groups were evaluated with Student t test. Levels of statistical significance for comparison of wt thymus versus wt LNs are *P < .02 and **P < .003 and for comparison of wt LN versus OM⁺ LNs, †P < .04, ††P < .008, and †††P < .001. Intracellular staining for Bcl-2 and phospho-Stat3 (P-Stat3) proteins was done (B,D,F) on wt thymus (solid black line), wt LNs (gray shaded histograms), and OM⁺ LNs (solid gray line) DN cells. Secondary antibody was used as a negative control for P-Stat3 staining (dotted line).