Fig.2|. Potentiation of Treg cell generation by isoDCA acts requires FXR expression in DCs.

(a) Effects of FXR deficiency in T cells (T^WT and T^ΔFXR) or DCs (DC^WT and DC^ΔFXR) on Treg cell induction by isoDCA (50 μM). (b-f) RNA-seq analysis of FACS-purified DCs 24 hours after BA exposure. (b) Transcriptional profiling of WT DCs treated with isoDCA (50 μM). Differentially expressed genes in orange. (c) Fold-change (FC) vs. FC plot comparing the transcriptional changes induced by isoDCA treatment (x-axis) and FXR deficiency (y-axis). Genes reduced by isoDCA in blue; genes induced by isoDCA in orange. (d) Overlap between genes regulated by isoDCA treatment (orange) and FXR deficiency (blue). (e) FC vs. FC plot comparing the effects of isoDCA on WT (x-axis) and FXR-deficient (y-axis) DCs, color-coded as in (c). (f) Overlap between genes regulated by isoDCA in WT (orange) and FXR-deficient (blue) DCs. (g) Differential scanning fluorimetry (DSF) experiment with recombinant FXR-LBD and BAs at 1000-fold (500 μM) or 200-fold (100 μM) excess. Showing the Boltzmann melting temperature (Tm). (h-i) Luciferase reporter assays. Cells expressing a Gal4-FXR-LBD fusion protein were treated with vehicle, CDCA, GW4064 alone or combined with isoDCA (100 μM). (j) FRET-based co-activator recruitment assay. BAs were mixed with GST-tagged FXR-LBD (5 nM), FITC-SRC2–2 coactivator peptide (500 nM) and Tb α-GST antibody (5 nM). Showing ratio of fluorescence at 520/485 nm. Displaying mean ± SD of technical replicates (a, g, n=3; j, n=4), representative of three independent experiments. Data in (b-f, n=3) are from one experiment. Data in (h-i, n=3) shown as mean ± SD, pooled from three independent experiments. Statistical significance determined by a one- (g) or two-way (a, h-j) ANOVA followed by Tukey or Sidak’s test. (*) p<0.05, (***) p<0.001, (****) p<0.0001 vs vehicle; (+) p<0.05 vs paired concentration of agonist; (ns) not significant.