## 2 Results
The pattern of inhibited/induced transactivation observed for the effects of these dual NR4A1/2 ligands in RKO cells was also observed in SW480 (Figure 3B) and HCT116 (Figure 3C) colon cancer cell lines and confirms that the CDIM compounds target both NR4A1 and NR4A2 in transactivation assays using GAL4-receptor chimeras but with inverse induction responses in colon cancer cells. A comparison of the effects of NR4A1 and NR4A2 knockdown and the DIM-3,5 dual NR4A1/NR4A2 ligands on colon cancer cell migration and apoptosis (TUNEL assay) was also investigated in SW480 cells. Compared to the untreated or scrambled non-specific oligonucleotide, siNR4A1 and siNR4A2 alone or in combination, with 7.5 or 15 umol/L DIM-3,5-CI\({}_{2}\), inhibited SW480 cell migration (Figure 4A). A comparable approach was also taken in SW480 cells using the TUNEL assay for apoptosis and both the knockdown of NR4A1 and NR4A2 and their combination and DIM-3,5-CI\({}_{2}\) induced TUNEL staining in SW480 cells (Figure 4B). Previous studies showed that both \(\beta\)1-integrin and G9a were NR4A1-responsive genes [23] and results in Figure 1 confirm that both genes are regulated by NR4A1 and NR4A2. It was previously reported that NR4A1 acted as a cofactor and NR4A1/Sp1 and NR4A1/Sp4 regulated \(\beta\)1-integrin and G9a, respectively, by binding their corresponding GC-rich promoter sequences. This mechanism where NR4A1 actsas a ligand-dependent cofactor to induce or repress gene expression has been previously observed for many other nuclear receptors [33]. Oligonucleotides targeting Sp1 (siSp1) and Sp4 (siSp4) were transfected into RKO and SW480 cells and both Sp1 and Sp4 decreased expression of G9a and \(\beta\)1-integrin in RKO and SW480 cells (Figure 5A). This suggested that Sp1 and Sp4 are also involved in the regulation of G9a, and \(\beta\)1-integrin expression and RKO cells were used as a model for further investigating the mechanisms of \(\beta\)1-integrin and G9a regulation. Figure 5B,C summarize the results of ChIP assays showing relative levels of NR4A1, NR4A2, Sp1 and Sp4 associated with an NR4A1-responsive GC-rich region of the \(\beta\)1-integrin promoter after treatment with DIM-3,5-CI\({}_{2}\). All four proteins are detected and there are some treatment-related changes such as decreased levels of NR4A2 and Sp1. The overall model (Figure 5C) shows interactions of both NR4A1 and NR4A2 with Sp1/4 on the \(\beta\)1-integrin promoter, and it is also possible that NR4A1 and NR4A2 bind Sp as monomers or homodimers, as previously suggested in studies on NR4A1 interactions with the \(\beta\)1-integrin promoter [34]. However, since it has previously been reported that NR4A1 and NR4A2 can also form heterodimers [35], it is possible that the NR4A1-NR4A2 heterodimer binds Sp1 or Sp4. Result obtained in a ChIP assay on the G9a promoter were similar to those obtained for \(\beta\)1-integrin; however, DIM-3,5-CI\({}_{2}\) treatment decreased interactions of NR4A1, NR4A2, Sp1 and Sp4 with the GC-rich region of the G9a promoter (Figure 5D). These results are consistent with the coregulation of G9a and \(\beta\)1-integrin by NR4A1 and NR4A2, and this complements the results of functional assays where both NR4A1 and NR4A2 exhibit pro-oncogenic activity that is inhibited by dual DIM-3,5 inverse NR4A1/NR4A2 agonists. In complementary experiments, the genomic effects of NR4A1 and NR4A2 were also investigated in SW480 cells using RNA interference and subsequent scRNA-seq analysis (Section 4). Figure 6A,B summarizes the effects of NR4A1/NR4A2 knockdown and DIM-3,5-CI\({}_{2}\) on NR4A1 and NR4A2 mRNA levels in SW480 cells. Figure 6C illustrates a UMAP visualization of the single cells according to their group/treatment identity. The results in Figure 6D,E show that DIM-3,5-CI\({}_{2}\) decreased the expression of NR4A1, c-parp, \(\beta\)1-integrin, G9a, Sp1 and Sp4 but increased NR4A2 mRNA levels in SW480 cells. DE analysis was performed using the default testing implemented in the R Seurat package (Section 4).