Ligands used to stimulate PBMCs (including cell signaling activators/inhibitors and receptor agonists/antagonists) were purchased from Sigma-Aldrich, Tocris/Bio-Techne, eBioscience/Affymetrix, R&D Systems, Life Technologies, Abcam, Antibodies-online, and Enzo Life Sciences. CHIR-99021 and JB1121 were synthesized in-house following published protocols (33). Table S2 lists the ligands used with their primary mechanisms of action and final assay concentrations (selected on the basis of published in vitro efficacy data and maximal clinical serum concentrations in the presence of serum proteins where relevant). Stock solutions of ligands were prepared in sterile conditions. Initial solubilization was achieved using DMSO where possible and, alternatively, PBS, H2O, or H2O with equimolar NaOH as per the manufacturer’s instructions. Intermediate dilutions were made in PBS, and DMSO was added to equivalent amounts for each ligand and vehicle.

The FDA-approved compound library (v.2.0; Enzo Life Sciences) was extended to incorporate experimental compounds contributed by collaborators [L. Jones-Brando, R. Yolken, G. H. Posner, J. G. D’Angelo, and C. P. Hencken (Johns Hopkins University School of Medicine, Baltimore, MD, USA); J. McNulty (McMaster University, Hamilton, Ontario, Canada); F. F. Wagner and E. B. Holson (Broad Institute of MIT and Harvard, MA, USA); T. L. Petryshen and S. J. Haggarty (Massachusetts General Hospital, Boston, MA, USA); and R. S. Williams (Royal Holloway University of London, Egham, UK)] and also positive controls/neutraceuticals, related to putative drug targets, from the CCNR compound library (table S8). Initial solubilization was conducted using DMSO where possible and, alternatively, PBS or H2O as per the manufacturer’s instructions. Intermediate dilutions were prepared using complete RPMI media without penicillin-streptomycin [RPMI-1640 with sodium bicarbonate (Sigma-Aldrich), 10% FBS (Life Technologies) to account for physiological serum protein binding of drugs, and 2 mM l-alanyl-l-glutamine dipeptide (Life Technologies)]. DMSO was added to equivalent amounts across all compounds and the vehicle. The final assay concentration of most of the compounds in the extended FDA-approved library was 20 μM with some exceptions in the compounds from collaborators and the CCNR library (table S8). All stocks and dilutions of stimulants and compounds were stored at −80°C, and repeated freeze-thaw cycles were avoided.

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