Journal: Cell Death and Differentiation

Article Title: Expression of Twist2 is controlled by T-cell receptor signaling and determines the survival and death of thymocytes

doi: 10.1038/cdd.2016.68

Figure Lengend Snippet: NFATc3-dependent Twist2 expression is repressed by JNK activity. (a) Thymocytes of DO-TCR-Tg mice were isolated at 3 h after i.p. injection with either PBS or OVAp (100 μg). Twist2 expression are presented relative to PBS-injected DO-TCR-Tg mice (error bars, ± S.E.M., n=4). (b) Thymocytes from Tcrα−/− mice were treated with P0.1/I200 or P10/I500 for the indicated time period (0, 1, 3, 6, and 9 h). Twist2 expression was measured by qPCR (error bars, ±S.E.M.). (c) 16610D9 cells were treated with I0/P0 (non), I200, P10/I200, or P10/I200+SP600125 for 3 h. Cells were stained with DAPI (blue) and anti-NFATc3 antibody, which was followed by staining with anti-rabbit-TRITC (red) antibody. (d) ChIP assay was performed by using anti-NFATc3 antibody after stimulation of 16610D9 cells with P0.1/I200 and P10/I500 for 0, 1, 2, and 3 h. The eluted supernatants as well as the Input extracts were PCR-amplified with primers for Twist2 promoter in the −185~+63 region

Article Snippet: Rag −/− × DO11.10 TCR-Tg mice were purchased from Taconic Farms (Germantown, NY, USA).

Techniques: Expressing, Activity Assay, Isolation, Injection, Staining, Amplification

Journal: Cell Death and Differentiation

Article Title: Expression of Twist2 is controlled by T-cell receptor signaling and determines the survival and death of thymocytes

doi: 10.1038/cdd.2016.68

Figure Lengend Snippet: Loss of Twist2 increases the sensitivity of thymocytes to TCR-mediated apoptosis. (a) The percentage of AnnexinV+ cells in TCRβ/CD69 subpopulations as in Figure 3c is summarized (error bars, ±S.E.M., n=6). (b) The percentage of DP cells in thymocytes obtained from DO-TCR-Tg mice was detected at 24 h after co-culture with OVAp (OVA323-339 peptide) pre-loaded B cells using FACS CantoII. The graph presents the relative percentage of DP cells to non-stimulated cells (error bars, ±S.E.M., n=6). (c) Indicated mice were crossed with Tcrα−/− mice to preclude TCR-experienced thymocytes. Apoptotic cells were detected after PMA and IM stimulation using AnnexinV staining kit. The graph presents the AnnexinV+ percentage in control and cKO mice (error bars, ±S.E.M., n=3)

Article Snippet: Rag −/− × DO11.10 TCR-Tg mice were purchased from Taconic Farms (Germantown, NY, USA).

Techniques: Co-Culture Assay, Staining

Journal: Cell Death and Differentiation

Article Title: Expression of Twist2 is controlled by T-cell receptor signaling and determines the survival and death of thymocytes

doi: 10.1038/cdd.2016.68

Figure Lengend Snippet: Twist2 inhibits TCR-mediated apoptosis and negative selection of thymocytes. (a) Indicated mice were crossed with Tcrα−/− mice. Apoptotic cells were detected after PMA and IM stimulation using AnnexinV staining kit. The graph presents the AnnexinV+ percentage in NLC and Twist2-Tg mice (error bars, ±S.E.M., n=3). (b) Representative CD4/CD8 and CD24/Vα2 FACS plots of the indicated mice. (c) The graph presents the total thymic cellularity in the indicated mice (left). Relative percentage of thymic cellularity in OT2-TCR × mOVA-Tg mice and OT2-TCR-Tg mice with either NLC or Twist2-Tg mice is shown (right) (error bars, ±S.E.M., n=3). (d) The percentage of the CD24loVα2hi population in total thymocytes from the indicated mice is summarized (error bars, ±S.E.M., n=3)

Article Snippet: Rag −/− × DO11.10 TCR-Tg mice were purchased from Taconic Farms (Germantown, NY, USA).

Techniques: Selection, Staining

Journal: Cell Death and Differentiation

Article Title: Expression of Twist2 is controlled by T-cell receptor signaling and determines the survival and death of thymocytes

doi: 10.1038/cdd.2016.68

Figure Lengend Snippet: Twist2 regulates the expression of Nur77 and Nor-1. (a–c) The expression level of Nur77 in the CD24loVα2hiCD4+ population was analyzed by flow cytometry using intracellular staining. Numbers indicate the percentage of Nur77hi cells in each population (blue solid line: OT2-TCR × mOVA-Tg, gray tinted line: OT2-TCR-Tg) (a). The percentage of Nur77hi cells (b) and the MFI of Nur77 (c) in the indicated mice are summarized (error bars, ±S.E.M., n=3). (d and e) The expression of Nur77 and Nor-1 was detected using qPCR in thymocytes from NLC and Twist2-Tg mice (d) or control and cKO mice (e) after stimulation with PMA and IM (PMA/IM). All mice were crossed with Tcrα−/− mice to preclude TCR-experienced thymocytes. The expression of Nur77 and Nor-1 expression is normalized to β-actin expression, and the results are presented relative to non-stimulated respective control thymocytes (error bars, ±S.E.M.). (f) Schematic diagrams of the Nur77 and the Nor-1 promoter constructs used in luciferase assay. Mutated sequences in each promoter are indicated in red color. (g and h) Twist2 expression vector was co-transfected into 16610D9 cells with Nur77 and Nur77-mutMEF2 promoter constructs (g) or Nor-1 and Nor-1-mutHDAC7 promoter constructs (h). Luciferase activities were detected after the PMA/IM treatment. The results are presented relative to the promoter activity in mock-transfected cells (error bars, ±S.E.M.). (i) ChIP assay was performed in thymocytes from DO-TCR × Twist2-Tg mice. The binding of Twist2 to the promoters of Nur77 and Nor-1 was detected by semi-qPCR. The band intensity was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to Input (error bars, ±S.E.M.)

Article Snippet: Rag −/− × DO11.10 TCR-Tg mice were purchased from Taconic Farms (Germantown, NY, USA).

Techniques: Expressing, Flow Cytometry, Staining, Construct, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Binding Assay, Software

Journal: Cell Death and Differentiation

Article Title: Expression of Twist2 is controlled by T-cell receptor signaling and determines the survival and death of thymocytes

doi: 10.1038/cdd.2016.68

Figure Lengend Snippet: 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.)

Article Snippet: Rag −/− × DO11.10 TCR-Tg mice were purchased from Taconic Farms (Germantown, NY, USA).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Binding Assay, Software, Western Blot, Construct, Luciferase, Staining