Fig 1: IOX1 regulated gene programs in Th17 cells. (a) Differentially Expressed Genes (DEG) changed by at least two fold with a P < 0.05 (DEseq2) in response to IOX1 treatment represented in Volcano plot. (b) Pathways enriched in IOX1 induced DEGs, analyzed using IPA. (c) Potential upstream regulators of IOX1 induced DEGs were analyzed using IPA. Th17 cells were collected at 72 h. The CETSA was performed to analyze the potential association between IOX1 and JMJD3, KDM3A (d) and between IOX1 and TET2, TET3 (e). (f) The response curve of TET2 and IOX1 in biolayer interferometry assay. The red rectangle highlights the changes identified in the CETSA of TET2.
Fig 2: IOX1 regulates Il17a expression through direct association with TET2 on the Il17a promoter. (a) DNA methylation status was assayed using bisulfite sequencing analysis of named CpG sites (#1–12) on the proximal promoter of Il17a in murine total CD4 polarized Th17 cells. A total of 36 clones from four independent experiments were sequenced. The methylation status of CpG site #7 and #8 were statistically different between DMSO and IOX1 treated Th17 cells (Fisher exact test, ∗P < 0.05). (b) Summary of the frequencies of demethylated CpG sites on the Il17a promoter. (c) Binding of TET2 on the CpG-5 and CpG-6-8 sites of Il17a promoter in response to IOX1 treatment, assayed by ChIP. (N = 3) (d) Binding of IOX1 to the CpG-5 and CpG-6-8 sites of Il17a promoter assayed by Chem-IP. (N = 2) (e) The chemical structure of biotinylated IOX1. (f) Representative FACS analysis of IL-17 expression in response to IOX1 (20 μM) in WT (CD4Cre) and TET2 deficient (CD4CreTet2f/f) Th17 cells. (g) Summary of IOX1 induced IL-17 suppression in WT (CD4Cre) and TET2 deficient (CD4CreTet2f/f) Th17 cells (N = 3). (h) The fold suppression of IL-17+ cell frequencies induced by IOX1 in WT (CD4Cre) and TET2 deficient (CD4CreTet2f/f) Th17 cells (N = 3). (∗P < 0.05, ∗∗P < 0.01, Mann–Whitney U test). (i) Summary of IOX1 induced suppression of CCL20 expression (in culture supernatants, measured by ELISA) in WT (CD4Cre) (N = 4) and TET2 deficient (CD4CreTet2f/f) (N = 3) Th17 cells. (j) The fold suppression of CCL20 expression induced by IOX1 in WT (CD4Cre) (N = 4) and TET2 deficient (CD4CreTet2f/f) (N = 3) Th17 cells. (∗P < 0.05, Mann–Whitney U test).
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