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Figure S2. Ectopic overexpression of T-bet does not induce twist1 gene expression in the absence of IL-12. Naive DO11.10 Th cells were stimulated twice under Th1-polarizing conditions (anti-IL-4, 5 ng/ml IL-12) or twice in the absence of IL-12 (anti-IL-4, anti-IL-12) to exclude twist1-inducing IL-12/STAT4 signaling from analysis. On day 2, the cells were infected with control virus or a virus encoding t-bet. Infected cells were isolated on day 12 according to expression of the viral marker gene gfp. (A) Twist1 mRNA was quantified by RT-PCR after activation for 3 h with PMA/ionomycin and IL-2. (B) Ectopic expression of T-bet was confirmed by immunoblot. Control: α-Tubulin immunoblot (bottom). Data are representative of two experiments.