Figure 5.

Loss of BATF impairs Th17 differentiation. (A) Rpkm values are plotted for BATF RNA at different time points of activation (Th0) or Th17-differentiation, using our published RNA-seq data (8). (B) Immunoblot (left) shows BATF protein levels in SCR versus BATF KD cells, at 24 h of Th17 polarization. Actin serves as loading control. Adjoining flow cytometry plots show percentage of CCR6 positive cells and the graph below shows ELISA analysis for IL-17 secretion in SCR versus BATF KD cells, at 72 h of Th17 polarization. ELISA values were first normalized for cell count (live), and then normalized to SCR control. Graph shows mean ± SEM for three biological replicates. Statistical significance was calculated using two-tailed Student's t test (**p < 0.01). (C) Volcano plots highlight the significantly upregulated (in red) and downregulated (in blue) genes in BATF-silenced Th17 cells at 24 h (left) and 72 h (right) of polarization (FDR ≤ 0.1, |FC| ≥ 1.8). DE genes with relevance to Th17 function are shown (extended DE gene list shown in Supplementary Figure S7D). (D) IPA was used to identify pathways altered upon silencing of BATF in Th17-polarized cells (24 h and 72 h). The top pathways related to T-cells and immune signaling are selectively shown. (E) Venn diagram shows the overlap between the genes that are altered upon BATF KD and the genes whose putative promoter regions (5-kb window around the TSS) are bound by BATF. The overlapping area represents the promoter-bound regulatory targets of BATF and the adjoining heatmap shows their corresponding expression changes in BATF KD Th17 cells. IGV images illustrate the occupancy of BATF over some of its Th17-associated targets. (F) Figure shows the topmost consensus sequence for genomic-binding of BATF, and the top six TF motifs enriched within BATF-bound sites, which were obtained using de-novo motif enrichment analysis by Homer. Peaks with IDR p value <0.01 were used for motif discovery.