Figure 4. KLF10 binds to the IL-9 promoter and interacts with HDAC to inhibit IL-9 activation.

A, Putative KLF10 transcription factor-binding sites in the mouse IL-9 promoter region. The putative binding sequences are highlighted. The transcription initiation site is defined as +1. B, Primary CD4+ T cells were isolated from Cre control mice and subjected to the chromatin immunoprecipitation (ChIP) assay with antibodies against IgG or KLF10. The immunoprecipitated DNA was subjected to semiquantitative PCR and q-PCR. C, The protein level of KLF10 after transfection. D-E, HEK293T cells were transfected with a full-length mouse IL-9 promoter luciferase reporter or 5’ truncations of the IL-9 promoter, together with expression plasmids encoding full-length mouse KLF10 or empty vector (mock control). F, Primary CD4+ T cells were treated with Ang II for 12h, then cells were harvested, lysed, and subjected to immunoprecipitation by the indicated antibodies. Immunoprecipitation (IP) were subjected to Western blotting with the indicated antibodies, and the quantification of IP. G-H, HEK293T were co-transfected with mouse KLF10 expression vector and the indicated mouse IL-9 promoter luciferase reporters (WT, Mut1, Mut2, or Mut3), and siRNA (non-specific or HDAC1), and the relative luminescence units (RLU) after transfection. B, and F, P values correspond to unpaired two-tailed Mann-Whitney U-tests. For normal distributed data, P values correspond to unpaired two-tailed t tests (D, and G), or two-way ANOVA with Tukey's multiple comparisons tests (E, and H). Ctrl indicates control; Ang II, angiotensin II; Luc, luciferase reporters; RLU, relative luminescence units.