Transcriptomic analysis of GM-CSF–derived human macrophages reveals a highly limited response to exogenous Ado and PGE₂. Donor matched human monocytes were cultured in M-CSF and GM-CSF for 7 days and were then left unstimulated or were stimulated with LPS, LPS + Ado, or LPS + PGE₂ for 4 hours before total mRNA was isolated and sequenced on an Illumina platform. (A) Coefficients of variation (CoV) were calculated and plotted for M-CSF (blue, 0.1) and GM-CSF (red, 0.088) samples stimulated with LPS, LPS + Ado, or LPS + PGE₂. The number of samples analyzed for M-CSF and GM-CSF are indicated above the violins (30 total). The y-axis represents the CoV, and the width of each violin changes according to the number of transcripts with that CoV. (B) Principal component analysis (PCA) indicates variance between samples and sample stimulations including LPS, LPS + Ado (LA) and LPS + PGE₂ (LP). (C) Volcano plots exhibit the log₂FC of all the genes expressed in macrophages stimulated by LPS + Ado and LPS + PGE₂ relative to macrophages stimulated by LPS alone for both M-CSF (top) and GM-CSF (bottom) samples. The numbers printed in the box are the number of DEGs with a fold change >2 and P < .05 (number of green dots). (D) The fold changes of the top 20 upregulated DEGs (n = 5; FC >2; P < .05) by LPS + PGE₂ and LPS + Ado in M-CSF macrophages (light blue bars) and their corresponding fold changes in GM-CSF macrophages (red bars) are plotted relative to LPS stimulation alone. (E) The fold changes of the bottom 20 downregulated DEGs (n = 5; FC >2; P < .05) by LPS + PGE₂ and LPS + Ado in M-CSF macrophages (light blue bars) and their corresponding fold changes in GM-CSF macrophages (red bars) are plotted relative to LPS stimulation alone. Note: CCL3L3 and CCL3L1 share the same ENSEMBL gene ID and were therefore represented together as 1 bar. *P ≤ .05; **P ≤ .01; ***P ≤ .001.