Figure 1.

Expression of Twist2 is induced by TCR signal via the Ca²⁺–Calcineurin–NFATc3 pathway in developing thymocytes. (a) The expression level of Twist2 was analyzed using intracellular staining by flow cytometry on the following populations of cells: DN (TCRβ^loCD69^loCD4⁻CD8⁻); pre-DP (TCRβ^loCD69^loCD4⁺CD8⁺); post-DP (TCRβ^int/hiCD69^hiCD4⁺CD8⁺); semimature SP (TCRβ^int/hiCD69^hiCD4⁺CD8⁻ and TCRβ^int/hiCD69^hiCD4⁻CD8⁺); and mature SP (TCRβ^hiCD69^loCD4⁺CD8⁻ and TCRβ^hiCD69^loCD4⁻CD8⁺). Gating strategy is described in Supplementary Figure S1a. Mean fluorescence intensity (MFI) of Twist2 is normalized to IgG, and the results are presented relative to DN stage (error bars ±S.E.M., n=9). (b) Twist2 expression in sorted pre-DP and post-DP cells was measured by quantitative PCR (qPCR). The expression of Twist2 is normalized to β-actin expression, and the results are presented relative to pre-DP stage (error bars, ±S.E.M., n=5). (c) 1 kb Twist2 promoter was transfected to 16610D9 cells, and the luciferase activities were measured after treatment with combinations with PMA (P0.1, 0.1 ng/ml; and P10, 10 ng/ml) and/or IM (I200, 200 ng/ml; and I500, 500 ng/ml). For those indicated below the graph, 5 μg/ml of CsA were treated 15 min before PMA and IM treatment. The graph presents the relative percentage of luciferase activities to P0.1/I200-treated cells (error bars, ±S.E.M., n=3). (d) Twist2 expression in thymocytes from Lck-Cre, CnB1^−/−, and NFATc1, c3^−/− mice was measured by qPCR after stimulation with P0.1/I200 for 3 h or not (non). Twist2 expression is normalized to β-actin expression, and the results are presented relative to not treated (non) thymocytes from Lck-Cre mice (error bars, ±S.E.M., n=3). (e) Nuclear extracts from 16610D9 cells were prepared 3 h after P0.1/I200 stimulation. Electrophoretic mobility shift assay was performed on the three putative NFAT-binding sites with the nuclear extracts. Before gel running, anti-NFATc3 antibody was incubated (+) or not (−). (f) 177 bp or NFAT-binding site mutants of Twist2 promoter constructs were co-transfected with a mock vector (mock) or a NFATc3 expression vector (NFATc3) in 16610D9 cells. After 36 h, cells were further activated with I200 for 6 h, and the luciferase activities were measured (error bars, ±S.E.M., n=3)