Figure 7.

Twist2 interacts with MEF2D and HDAC7 to repress the expression of Nur77 and Nor-1. (a) Twist2, Twist2CΔ, MEF2D, and HDAC7 were cloned into either VN-173 or VC-155 vector. Indicated VN/VC vector combinations were transfected into 293T cells, and fluorescence was detected after 48 h using fluorescence microscope (LSM 710). (b and c) 16610D9 cells were transfected with either a mock or a Twist2 expression vector. The binding of HDAC7 (b) or p300 (c) was detected by ChIP assay after PMA and IM stimulation. The binding of HDAC7 and p300 was quantified using the ImageJ software and normalized to Input. The graphs present the relative bindings to non-stimulated mock (error bars, ±S.E.M.). (d) HDAC7 was detected in the nuclear extract (NE) in sorted OT2-TCR⁺ DP thymocytes obtained from OT2-TCR-Tg mice and OT2-TCR × Twist2-Tg mice by western blotting. (e) The Nur77 promoter construct was co-transfected with either a Twist2 or a Twist2CΔ vector in 16610D9 cells. Transfected cells were activated 1 h with PMA/IM or not treated (non). Relative luciferase activities to PMA/IM-treated mock are shown (error bars, ±S.E.M.). (f) 16610D9 cells were transfected with either a Twist2 or a Twist2CΔ vector and apoptotic cells were detected using AnnexinV staining after PMA and IM stimulation for 6 h. The graph presents the relative percentage of AnnexinV⁺ cells to mock-transfected cells (error bars, ±S.E.M.)