Fig 1: Testosterone induces PI3K pathway alterations with adipocytes showing distinct metabolic adaptations(A) Heatmap showing the log2 fold change in RNA expression of PI3K activating receptors (taken from "KEGG: PI3K-Akt signaling pathway") among 10 breast cell types identified. The circle diameter indicates the fraction of cis-female cells of the cell type expressing the receptor. INS, circulating insulin secreted in the pancreas.(B) Heatmap showing log2 fold changes in RNA expression of KEGG: PI3K-Akt signaling pathway downstream transcription factors within 10 breast cell types identified.(C) Pathway enrichment of tissue wide upregulated genes (>7 cell types) that have an NR4A1 motif in an enhancer (>4 cell types). Horizontal axis shows the odds ratio (one-sided Fisher’s exact test) comparing frequency of selected genes in the pathway versus background and vertical axis shows –log10 p value of Fisher’s exact test (FDR < 0.05 = red, p value < 0.05 = yellow, n.s. = gray (PID, Pathway Interaction Database; WP, WikiPathways).(D) Module scores for the WikiPathways "WP: insulin signaling pathway" in all cell types, split by cis-female and trans-male origin (p value in adipocytes, Wilcoxon: 7.59 × 10-4).(E) Representative images of computational segmentation of lipid vacuoles (left, see STAR Methods), and resulting average area of adipocyte vacuoles per IHC scan region (p value Wilcoxon: 0.00059).(F) Sample averages of AZGP1 RNA expression in each cell type in trans-male and cis-female samples (adjusted p values, MAST: adipocyte = 5.95 × 10-12, basal = 2.42 × 10-70, blood EC = 8.67 × 10-83, fibroblast = n.s., luminal-HR- = 4.54 × 10-302, luminal-HR+ = 0.00, lymph. EC = 2.83 × 10-15, lymphoid cells = 5.46 × 10-12, myeloid cells = 9.23 × 10-8).(G) Microscopic CODEX image of a duct stained against AR (red), AZGP1 (green), and ACTA2 (purple) in a trans-male (top) and cis-female (bottom) breast tissue region of the tissue microarray.(H) GPAM co-expression module (GRNboost2, 95th percentile, p value, Wilcoxon: <2.22 × 10-16) score in cis-female and trans-male adipocytes.(I) TCF7L2 expression in trans-male and cis-female adipocytes (adjusted p value, MAST: 3.48 × 10-105).(J) Differential expression of transcription factors in trans-male compared with cis-female adipocytes. Horizontal axis shows log2 fold change in expression and the vertical axis shows –log10 adjusted p value. Purple data points indicate transcription factors with accessible chromatin matching the AR sequence motif (CisBP AR_689).(K) Microscopic IHC image showing staining against nuclei (DAPI, blue), adipocytes (PLIN1, green), and TCF7L2 (purple). Boxplot shows the median staining intensity of TCF7L2 among IHC scan regions of cis-female and trans-male adipocytes (p value, Wilcoxon: 0.0069).