Extended Data Figure 3. Weighted Gene Co-expression Network Analysis (WGCNA) identifies distinct and coordinate gene expression patterns in the fasting and refed states.

a, Modules of genes with correlated expression patterns were clustered using WGCNA. The modules, with their distinct color designations using the fasted and refed data are shown on the x and y axes. Numerical assignment aligned to module colors represent number of genes per module. The correlated modules from fasting and refed were aligned to determine gene overlap (significance of the overlap determined by Fisher’s exact test where red shading denotes significance - darker shade > significance). b, Representative cell type enrichment analysis (CTen) result of a fasting cluster showing enrichment of CD8⁺ and CD4⁺ T cells encoding genes (p-value depicted by red geometric plot extending from the center of the figure towards enriched cell types – representing −log10 Benjamini-Hochberg adjusted P. c, GeneMANIA derived protein-protein interaction (PPI) networks. Significant DE genes fold change information where blue circles represent increased, and red circles decreased expression during fasting compared to refeeding. d, qRT-PCR validation of selected network genes. Bar graph represent mean±SEM with value of each subject (n=14 subjects following 3–4 replicates using two-sided paired Student’s t-test). e, Relative FOXO4 RNA expression in CD4⁺ T cells in response to siRNA (Bar graph represent mean±SEM, n=6 health volunteers, two-sided, paired Student’s t-test). f, Representative protein blot show FOXO4 expression in FOXO4-siRNA treated CD4⁺ T cells from healthy subjects (n=3), 3 days following TCR activation (+). g-h, Representative immunoblots showing expression levels of (g) FOXO4- and (h) FKBP5-overexpression in CD4⁺ T cells from healthy subjects (n=3). Open arrowheads - endogenous protein bands, overexpressed tagged-proteins - solid arrowheads. Source Data Extended Data Fig.3: Unprocessed immunoblots.