Fig 2: CyC is a glucocorticoid response gene in vitro(A) Co-localization of summary statistics for CyC-production from UKB and plasma cortisol from CORNET Consortium at SERPINA1/6 locus. rs2749527 variant is highlighted in red.(B) Trans-eQTL analysis examining association between genetic instrument rs2749527 and CST3 gene expression in visceral adipose fat (VAF) in STARNET cohort.(C) Cis-eQTL association between rs2749527 and SERPINA6 (encodes cortisol-binding globulin) in liver in STARNET cohort. p values for additive and recessive models are shown. See Figure S3 for replication analysis in GTEx.(D) Gene set enrichment analysis (MAGMA) across CyC-production summary statistics (UKB) for steroid signaling-related gene sets.(E) Functional genomics in A549 cell line (ENCODE project) treated with 100 nM dexamethasone for 0 min to 12 h. ChIP-seq (for glucocorticoid receptor/NR3C1) and ATAC-seq (at 0 h) at CST3 locus identifies a glucocorticoid-responsive and accessible distal enhancer element.(F and G) Time course of (F) GR recruitment (at distal enhancer) and (G) CST3 gene expression (log-CPM) following dexamethasone (DEX) treatment in A549 cells (ENCODE project). Trendline and shaded 95% confidence interval correspond to regression of gene expression as a function of log-time.(H and I) Extracellular CyC concentration in (H) A549 cells and (I) HeLa cells normalized to cellular protein content after 18-h treatment with 100 nM DEX or vehicle (VEH) control. Each condition comprises at least 5 biological replicates; horizontal bars indicate mean extracellular CyC. p values correspond to two-sided t tests. See Figure S3 for timecourse.(B and C) Boxplots show median (central line) with IQR (box) and extrema (whiskers at 1.5× IQR). Outliers beyond 1.5× IQR are shown as dots. SH, steroid hormone; SP, signaling pathway; VEH, vehicle; DEX, dexamethasone.

Fig 3: CyC is predominantly produced by myeloid cells in health(A) Tissue-specific expression quantitative trait score (eQTS) analysis to identify tissues with significant correlation (Spearman coefficient) between CyC-production PGS and tissue-specific CST3 gene expression in GTEx cohort. p values are uncorrected, as each correlation test is performed in a non-overlapping set of tissue-specific samples.(B and C) Distribution of normalized single-cell CST3 expression (log-transcripts per million [TPM]) in cell clusters isolated from (B) spleen and (C) peripheral blood mononuclear cells (PBMCs). Clusters defined by correlation to reference PBMC data.60(D) Mean CST3 gene expression (log-TPM) in each cell cluster from multiple tissue-specific single-cell RNA sequencing projects, harmonized by Human Protein Atlas. The top cell cluster and tissue-specific macrophage cell type (if not top cluster) by tissue is annotated.(E) Two-sample Mendelian randomization using blood-specific cis-eQTLs for CST3 (eQTLGen) as exposure and CyC-production latent trait GWAS as outcome. Error bars correspond to standard errors, and point color refers to linkage with top cis-eQTL.(F and G) Non-significant (p > 0.05) changes in (F) CST3 gene expression (reverse transcription PCR) during 0- to 18-h DEX (100 nM) treatment and (G) extracellular CyC concentration in human THP-1 cells (monocyte-like) normalized to cellular protein content after 18-h treatment with DEX (100 nM) or VEH control.(H and I) Each condition comprises 10 biological replicates. Plasma CyC concentration in healthy (H) BALB/cJ and (I) C57BL/6J mice treated with VEH or 20 mg/kg DEX.(J) Creatinine-normalized plasma CyC (C2 ratio) in patients with primary adrenal insufficiency treated with placebo (VEH) or hydrocortisone (CORT) in a crossover experimental medicine study. The administered intranveous (i.v.) CORT dose was 0.03 mg/kg/h between 12 and 7 a.m. (the time point of sampling), achieving near-physiological GC exposure.(G–J) p values refer to two-sided t tests. VEH, vehicle; DEX, dexamethasone.

Fig 4: CyC production is dynamically regulated in disease states(A and B) Significant (p < 0.05) changes in paired (A) CST3 gene expression (reverse transcription PCR) and (B) extracellular CyC concentration in PMA-treated human THP-1 cells (macrophage-like) normalized to cellular protein content during 0- to 18-h DEX (100 nM) treatment. There are 6 biological replicates per group.(C) Change in normalized extracellular CyC concentration in macrophage-like THP-1 cells after 18-h treatment with DEX (100 nM) or VEH control.(D and E) Creatinine-normalized plasma CyC (C2 ratio) at specific time points with sufficient data in hospitalized COVID-19 patients treated with DEX or standard of care (control [CTRL]) as part of cohorts based in (D) Calgary, Canada, and (E) Charité Hospital, Germany. Day 1 in the Calgary, Canada, cohort refers to a time window of 72 h after admission to the ICU. Error bars indicate standard error of the mean.(F) Single-cell CST3 gene expression in each cell cluster in melanoma tumors (n = 12) from Jerby-Anon et al.64 Clusters defined by correlation to reference PBMC data,60 with unclassified cells that exhibit detectable clonal copy-number variation classified as tumors.(G) Plasma CyC concentration in BALBc mice after inoculation with colon-26 (C26) tumor cells. Cachexia is defined by >15% body weight loss, and pre-cachexia refers to 14 days after tumor inoculation; tumor bearing refers to day 7 after tumor inoculation.(H) Significant positive correlation between plasma corticosterone and plasma CyC during tumor progression in C26 model.(I) Extracellular CyC concentrations in C26 cells normalized to cellular protein content after 0-, 6-, 12-, 18-, or 24-h treatment with 100 nM DEX. Each time point comprises at least 4 biological replicates. p values refer to two-sided t tests. VEH, vehicle; DEX, dexamethasone.