Fig. 1: Generation of a de novo steroidogenesis reporter and conditional knockout mice.
From: Tumors induce de novo steroid biosynthesis in T cells to evade immunity
a. Schematic of de novo steroidogenesis pathway.  Cyp11a1 is the first and a key rate-limiting enzyme of the pathway. b. Cyp11a1-reporter mice synthesize a fusion protein that self-cleaves due to presence of a T2A peptide and dissociates into Cyp11a1 and H2B-mCherry. c Cyp11a1-mCherry reporter mice report Cyp11a1 expression accurately. Single-cell suspensions of tissues and naïve splenic CD4+ T cells were analyzed by flow cytometry. Gating: All cells>Singlets>Live cells>Cyp11a1-mCherry. Representative of three independent experiments; each experiment contains 3–4 mice. d, e Generation of a Cyp11a1 conditional knockout (cKO) mice. Schematic presentation of the The targeting allele (d) and T cell-specific Cyp11a1 cKO (Cd4−Cre;Cyp11a1fl/fl) generation (e). f Cyp11a1 knockout efficiency of Cre recombinase in T cells. Splenic naïve T helper cells from cKO (Cd4-Cre;Cyp11a1fl/fl) mice or control mice (wild type and Cd4-Cre) were activated under Th2 differentiation condition, and analyzed for Cyp11a1 protein expression by western blot. TATA-binding protein (TBP) used as loading control. g Normal thymic development of T cells in Cyp11a1 cKO. Thymus was harvested from Cyp11a1 cKO and control (Cd4-Cre and Cyp11a1fl/fl) mice, dissociated into single-cell suspension, stained with fluorescent conjugated anti-CD4, CD8, B220, CD45, CD25, and CD44 antibodies, and analyzed by flow cytometry. Gating: all cells>Singlets>Live cells>CD45+B220−>CD4, CD8 (left panel). CD4+CD8+ cells represent double positive (DP) stage, CD4+CD8− and CD4−CD8+ cells represent single positive (SP) stage, CD4−CD8− cells represent double negative (DN) stage. DN cells of the left panel were gated to show CD25 and CD44 expression to identify DN1 (CD25−CD44+), DN2 (CD25+CD44+), DN3 (CD25+CD44−), and DN4 (CD25−CD44−). Error bars represent mean with s.d., N = 4 biologically independent animals. (h, i). Flow cytometric analysis shows normal distribution of Cyp11a1 cKO T cells (CD4+ and CD8+) in the peripheral blood and spleen. Error bars represent mean with s.d., N = 4 biologically independent animals.
