Supplementary Figure 5: Analysis of JunD and BACH2 binding in d5 in vitro–activated CD8⁺ T cells.

a, Naïve CD8⁺ T cells were isolated by flow cytometric sorting from spleens of wildtype animals and stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 2 days followed by 3 further days of culture to generate d5 in vitro activated CD8⁺ T cells. Protein expression was measured at indicated timepoints following stimulation using SDS-PAGE and immunoblotting. Quantification of BACH2 abundance normalized to β-actin is shown. BACH2 expression in day 5 in vitro activated CD8⁺ T cells is ~25% of that in naïve cells. b, Histogram of JunD binding, centered around JunD peaks, in WT and Bach2 KO CD8⁺ T cells at sites bound exclusively by JunD (left histogram) or sites at which JunD and BACH2 binding sites colocalize (right histogram). A significant increase in JunD binding is observed in Bach2-deficient cells only at sites where BACH2 and JunD binding sites colocalize. c-d, Analysis of exclusive JunD binding in Bach2-deficient cells. Pie chart showing JunD binding sites in Bach2-deficient cells (c). A majority of sites (blue) were found to be shared with WT cells, while a minority of sites (red) were only present in Bach2 KO cells. Alignments of JunD binding in WT and Bach2 KO cells at loci where exclusive JunD binding in Bach2 KO cells is detected (d). Arrows indicate loci at which JunD peaks only pass a low significance threshold for peak calling in Bach2-deficient cells (p<1x10^-3; Binomial test).