## 2 Results
### Comparison of \(CO_{2}\) Responsiveness in Cells Depleted of NR4A2 and NR4A3  
The data from Figure 2 indicates a significant overlap in transcripts affected by elevated CO\({}_{2}\) in both non-target cells and shRNA2 cells. Commonly regulated genes include those associated with mitochondrial gene expression, e.g., Mitochondndrially Encoded Cytochrome C Oxidase 1 (_MT-CO1_), Mitochondndrially Encoded NADH: Ubiquitinne Oxidoreductase Core Subunit 4 (_MTND-4_), Mitochondndrially Encoded NADH: Ubiquitinne Oxidoreductase Core Subunit 4L (_MT-ND4L_) (Supplementary Figure S6) and genes involved in cellular metabolism Pyrroline-5-Carboxylate Reductase 1 (_PYCR1_) (Figure 4A,C). Genes associated with immune signalling, e.g., Tumour Necrosis Factor (_TNF_) (Figure 2C and Supplementary Figure S7B) and _NR4A3_ (Figure 2D and Supplementary Figure S9C) were selectively CO\({}_{2}\) sensitive in shNR4A2 cells. Similarly, the data from Figure 3 indicates a significant overlap of transcripts affected by CO\({}_{2}\) in both non-target cells and shNR4A3 cells. Commonly regulated genes include those associated with cellular metabolism (_PYCR1_) (Figure 4A,C); however, the signature for mitochondrial and metabolism-associated genes is less striking in shNR4A3 cells (Supplementary Figure S8). Genes associated with immune signalling, e.g., _NR4A1_ (Figure 3C, Supplementary Figure S9A) and Signalling Lymphocytic Activation Molecule Family Member 1 (_SLAMF1_) (Figure 3D, Supplementary Figure S4B) were selectively CO\({}_{2}\) sensitive in shNR4A3 cells. Figure 2: Hypercapnia regulates gene expression in _NR4A2_-depleted monocytes in the presence and absence of LPS. Volcano plot of differential expression between 5% and 10% CO\({}_{2}\) (4 h) in shNR4A2 THP-1 cells in the absence (**A**) and presence (**B**) of LPS (2.5 ug/mL for 2 h). The cut-off for significance (adjusted \(p\)-value (\(p\)-adj) \(<\) 0.05) is shown with a solid line on the \(y\)-axis. 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 shRNA2 (orange) 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. From our comparisons of the CO\({}_{2}\) sensitivity of shNR4A2 cells and shNR4A3 cells compared to shNT cells, we observed several genes whose response to hypercapnia was conserved in _NR4A2_ or _NR4A3_-deficient cells, as well as genes that were uniquely sensitive to CO\({}_{2}\). Examination of RNA-sequencing (RNA-seq) transcript per million (TPM) data from the uniquely sensitive transcripts revealed that while some transcripts failed to reach the threshold for CO\({}_{2}\) sensitivity, the mean trends were qualitatively similar to those observed in the other cell types, e.g., _TNF_ is selectively decreased in response to CO\({}_{2}\) in the basal state in shNR4A2 cells; however, a decrease is also observed in shNT cells and shNR4A3 cells (albeit non-significantly) (Supplementary Figure S7B). Similarly, there were transcripts that were clearly affected by shNR4A2 on a basal level, e.g., Wnt Family Member 7B (_WNT7B_), but retained an equivalent level of CO\({}_{2}\) sensitivity (Supplementary Figure S7A). Thus, the data generated to date indicates that NR4A family members _NR4A1_-3 are CO\({}_{2}\)-sensitive transcripts in monocytes and selectively modulate elements of the monocyte response to buffered hypercapnia without being master regulators. Figure 3: Hypercapnia regulates gene expression in NR4A3-depleted monocytes in the presence and absence of LPS. Volcano plot of differential expression between 5% and 10% CO\({}_{2}\) (4 h) in shNR4A3 THP-1 cells in the absence (**A**) and presence (**B**) of LPS (2.5 ug/mL for 2 h). The cut-off for significance (\(p\)-adj) \(<\) 0.05) is shown with a solid line on the \(y\)-axis.