Figure 3.

Ingenuity pathway analysis of pregnenolone sulfate-induced differentially regulated genes. (a) IPA canonical signaling pathway enrichment analysis of differential expression of the pregnenolone sulfate treatment effect at the 24 h time point. The strength of inhibition or activation of each signaling pathway is shown in colors corresponding to IPA activation/inhibition z-scores. Dark orange and dark blue indicate strong activation and inhibition predictions, respectively. (b) Western blot analysis of whole-cell lysates from expanded T cells of one donor that were TCR-activated and either non-treated, or treated with 200 μM of pregnenolone sulfate (PS) for 2, 8, or 24 h, run on a gel and blotted with CHOP and GAPDH antibodies. The original blots are presented in Supplementary Figure S10 (c) Bar graph showing the mean fluorescence intensity (MFI) of the Mito-HE dye staining expanded T cells from three donors (represented by the different shapes) that were activated with CD3/CD28 beads for 24 h and either non-treated, treated with 250 μM of TBHP for 1 h or with 200 μM of PS for 2 h. (d) Extracellular flux analysis with the graph showing oxidative consumption rate of expanded T cells from three donors that were activated with CD3/CD28 beads for 24 h, followed by no treatment or treatment with 200 μM of PS for 24 h and subjected to the ATP rate test. (e) Same ATP rate test as in (d) showing the mitochondrial (dark colors) and glycolytic (light color) ATP production rate from non-treated and treated T cells (individual donors represented by different shape). Paired t-test was performed on (c) and (e); *p < 0.05, **p < 0.01, ***p < 0.001. (f) Summary illustration of the effect of pregnenolone sulfate on the T cell transcriptome and effector functions.