(A) Venn diagram displaying the number of DEGs (1.5-fold, up or down; P < 0.05) identified by RNA-Seq assays in Th17-polarized RORγt-expressing RORγt^–/– CD4⁺ T cells (RORγt throughout) compared with RORγt^–/– cells (EV) and in IMU-935–treated (500 nM) compared with nontreated RORγt⁺ CD4⁺ T cells. (B) Volcano plots displaying the global gene expression changes. The horizontal dotted line marks P = 0.05; the vertical dotted lines mark fold change of ±1.5. (C) The horizontal bars denote pathways identified by IPA canonical signaling pathways analysis. Upregulation (z > 0) or downregulation (z < 0) of the pathway activity in RORγt⁺ versus RORγt^–/– cells (orange) or IMU-935–treated compared with nontreated RORγt⁺ cells (blue). The vertical dotted lines mark P = 0.05. (D) Heatmap showing Th17 signature gene expression in RORγt^–/– (EV) or RORγt⁺ CD4⁺ cells treated with IMU-935 or not. Right: The gene set enrichment plot showing enrichment of Th17 genes. Gene expression was normalized by total counts of each gene. Specific Th gene sets were derived from the MSig database. (E) Heatmap depicting changes in the activity of the upstream regulators predicted by IPA. Activation z score indicated increased (orange; z > 0) or decreased (blue; z < 0) activity, all z score values are labeled within the corresponding cell. (F) Genome-wide RORγt-DNA binding signal intensity determined by ChIP-Seq assays near the TSS in RORγt^–/– (EV) or IMU-935–nontreated (–) or treated (+) RORγt⁺ CD4⁺ cells polarized under Th17 conditions for 3 days. (G) RORγt binding peaks at Il17a–Il17f (left) or Il23r (right) gene locus. The results are the representative of overlays from 2 separate experiments. NES, normalized enrichment score.