Figure 3. mitoROS is necessary and sufficient for isoalloLCA-dependent enhanced expression of FoxP3.

a, Chemical structures of LCA and its isomers: isoLCA, alloLCA and isoalloLCA. b, FoxP3 expression from mouse naïve CD4⁺ T cells cultured for 3 days with anti-CD3/28 and IL-2. DMSO or bile acids at 20 μM were added to cell culture (n = 3/group). c, qPCR analysis for FoxP3 transcripts in DMSO- or isoalloLCA- (20 μM) treated cells (n = 3/group). d, Diagram of the FoxP3 gene locus containing the promoter region (Pro) and intronic enhancer regions (CNS1, CNS2 and CNS3). e and f, Flow cytometric analyses and quantification of CD4⁺ T cells stained intracellularly for FoxP3. Naïve CD4⁺ T cells isolated from wild-type control, CNS1, CNS2 or CNS3 knockout mice were cultured with anti-CD3/28 and IL-2, in the presence of DMSO or isoalloLCA (20 μM) (n = 3/group). g, Mitochondrial ROS production measured by mitoSOX staining with T cells cultured in the presence of DMSO or LCA isomers for 48h. Staining intensity was reported as mean fluorescence intensity from flow cytometry analysis (PE channel). Different conditions were then normalized as fold change to the values of DMSO condition (n = 3/group). h and i, Representative FACS plots and quantification of T cells stained intracellularly for FoxP3, cultured with anti-CD3/28, IL-2 and TGF-β (0.05 ng/ml) in the presence of DMSO, LCA, isoalloLCA (20 μM) or retinoic acid (1 nM), with DMSO or mitoQ (0.5 μM) for 72 h (n = 3/group). j and k, Flow cytometric analyses and quantification of CD4⁺ T cells stained intracellularly for FoxP3. Naïve CD4⁺ T cells isolated from control or CNS3 knockout mice were cultured with anti-CD3/28 and IL-2 in the presence of DMSO or mitoPQ (10 μM) (n = 3/group). n, number of biologically independent samples. Data are shown as the mean ± standard deviation by unpaired t-test with 2-tailed p-value.