## 2 Results
### Comparison of \(CO_{2}\) Responsiveness in Cells Depleted of NR4A2 and NR4A3
Significantly upregulated genes are shown in green, and downregulated genes are shown in red. Venn diagram representing the number of genes differentially expressed in hypercapnia (4 h) in shNT (blue) and shNR4A3 (green) THP-1 cells in the absence (**C**) and presence (**D**) of LPS (2.5 ug/mL for 2 h). Data is representative of \(n=3\) individual experiments with examples included for each group. Many Genes Are Commonly Regulated by CO\({}_{2}\) in shNT, shNR4A2, and shNR4A3 Cells  
We next exploited the similarities in the responses of our three different cell lines to elevated CO\({}_{2}\). We reasoned that transcripts that were consistently CO\({}_{2}\) sensitive across three genetically modified THP-1 cell lines (with very different basal transcription of 100s of genes due to shNR4A2 or shNR4A3 [15]) represent particularly robustly CO\({}_{2}\) responsive transcripts. These data relating to core CO\({}_{2}\)-responsive genes can enhance our understanding of the cellular response to hypercapnia in monocytes with and without LPS treatment. In the basal state, 27 genes were commonly differentially expressed across all 3 groups (Figure 4A). These included genes involved in cellular metabolism (_PYCR1_, Aldehyde dehydrogenase 1 Family Member B1 (_ALDH1B1_)), regulation of calcium (Cluster of Differentiation 38 (_CD38_), Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase1 (_ATP2A1_), and serpin peptidase inhibitors Serpin Family B Member 2 (_SERPINB2_) and Serpin Family H Member 1 (_SERPINH1_)). The mitochondrial oxidative phosphorylation (OXPHOS) genes (_MT-CO1_, _MT-CO2_, _MT-CO3_, _MT-ND4_, _MT-ND4L_, and _MT-ND5_) were significantly differentially expressed in hypercapnia in shNT and shNR4A2 cells, but not in shNR4A3 cells in the basal state. In the presence of LPS, there were 360 common differentially expressed genes (DEGs) across all 3 groups (Figure 4B). This included genes involved in inflammation (Nucleotide-Binding Oligomerisation Domain, Leucine Rich Repeat and Pyrin Domain Containing 3 (_NLRP3_)), calcium signalling (Calcium/Calmodulin Dependent Protein Kinase II Gamma  
Figure 4: Comparison of CO\({}_{2}\)-sensitive genes in shNT, shNR4A2, and shNR4A3 THP-1 monocytes. Venn diagram representing the number of genes differentially expressed in hypercapnia (4 h) in shNT (blue), shNR4A2 (orange), and shNR4A3 (green) THP-1 cells in the basal state. Representative examples are included for each group (**A**). Venn diagram representing the number of genes differentially expressed in hypercapnia (4 h) in shNT (blue), shNR4A2 (orange), and shNR4A3 (green) THP-1 cells in the presence of LPS (2.5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for 2 h). Representative examples are included for each group (**B**). Venn diagram representing the number of genes differentially expressed in hypercapnia (4 h) in shNT/shNR4A2/shNR4A3 THP-1 cells in the basal (grey) and LPS stimulated (purple) (2.5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for 2 h) states. Representative examples are included for each group (**C**). A forest plot represents the differential expression of 9 genes that were CO\({}_{2}\) responsive regardless of NR4A expression and LPS stimulation (**D**). Data is representative of \(n=3\) individual experiments. (CAMK2G), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha-2/Delta-3 (_CACNA2D3_)) and solute carriers (e.g., Solute Carrier Family 6 (Neurotransmitter Transporter, Taurine), Member 6 (_SLC6A6_), Solute Carrier Family 16 (Monocarboxylic Acid Transporters), Member 9 (_SLC16A9_), Solute Carrier Family 17 (Acidic Sugar Transporter), Member 5 (_SLC17A5_), Solute Carrier Family 39 (Metal Ion Transporter), Member 8 (_SLC39A8_), Solute Carrier Family 39 (Metal Ion Transporter), Member 14 (_SLC39A14_)).