## 2 Results
DE genes were identified by comparing the DMSO control group versus each of the other groups, including transfection with siRNAs individually targeting NR4A1 or NR4A2, versus combined NR4A1 and NR4A2 siRNAs or treatment with DIM-3,5-CI\({}_{2}\). Overlap of identified up- and downregulated DE genes between different comparison pairs is shown in Venn diagrams (Figure 7A,B). The numbers of overlapping DE genes between different pairwise group comparisons were small, ranging from 14 (downregulated DE genes shared between control versus DIM-3,5-CI\({}_{2}\) and control versus siNR4A1) to 77 (unregulated DE genes shared between control versus DIM-3,5-CI\({}_{2}\) and control versus siNR4A2) out of the top 500 genes in each group. The limited overlap suggests relatively independent effects at the individual gene level in the siRNA knockdown and DIM-3,5-CI\({}_{2}\) treatments in SW480 cells. However, at the pathway level, the results of functional enrichment analysis with these DE genes were converged to several specific pathways and gene ontology terms, including, for example, _Apoptotic Signaling Pathway_, _Epithelial Cell_ or _Fibroblast Proliferation_ and _MAP Kinase Activity_ (Figure 7C). Thus, the pathway-level analysis suggests that the siRNA system and DIM-3,5-CI\({}_{2}\) treatment produced a similar functional impact on gene expression programs in SW480 cells. Figure 1: DIM-3,5 analogs inhibit cancer cell growth and modulate expression of NR4A1-regulated gene products. RKO (**A**), SW480 (**B**) and HCT116 (**C**) cells were treated with 2.5–20 \(\upmu\)mol/L DIM-3,5-Cl\({}_{2}\), DIM-3-Cl-5-CF\({}_{3}\) and DIM-3,5-Br\({}_{2}\) for 24 h and effects on cell proliferation were determined as outlined in Section 4. RKO and SW480 cells were treated with DIM-3,5 analogs for 24 h and whole-cell lysates were analyzed by Western blots (in triplicate), G9a, \(\beta\)1-integrin and apoptosis (**D**,**E**) and Sp/NR4A gene products (**F**,**G**). Results are expressed as means \(\pm\) SE for at least 3 determinations for each treatment group and significant (\(p<0.05\)) inhibition/induction compared to DMSO (control) is indicated (*) **(A–C)**; quantitative results for (**D–G**) are summarized in (**H**,**I**) within the figure. Figure 2: NR4A1 and NR4A2 knockdown on colon cancer cell growth and expression of selected genes. RKO, SW480 and HCT116 cells were transfected with oligonucleotides targeting NR4A1 (siNR4A1) and NR4A2 (siNR4A2) (**A–C**) and cell viability was determined. Multiple oligonucleotides targeting NR4A1 and NR4A2 and their combination were transfected into RKO or SW480 cells and whole-cell lysates were analyzed by Western blots knockdown to determine efficiency (**D**) and effects on selected gene products (**E**) as outlined in Section 4. Significant (\(p<0.05\)) inhibition is indicated (*) in (**A–C**). Figure 4: NR4A1 and NR4A2 knockdown and DIM-3,5 analogs inhibit invasion and induce TUNEL staining. SW480 cells were transfected with siRNA1 and siRNA4A2 or treated with DIM-3,5 analogs, and migration (**A**) and TUNEL. (**B**) staining were determined as outlined in the Section 4 Results are expressed as means \(\pm\) SE for at least 3 determinations per treatment group, and significant (\(p\) < 0.05) differences from the control group are indicated (*). Figure 3: Effects of DIM-3,5 analogs on receptor-dependent transactivation. RKO (**A**), SW480 (**B**) and HCT16 (**C**) cells were transfected with GAL4-NR4A1 or GAL4-NR4A2 and a UAS-luc reporter gene and treated with DIM-3,5 analogs and luciferase activity was determined as outlined in Section 4. Results are expressed as means \(\pm\) SE for at least 3 replicate determinations per treatment group, and significant (\(p\) < 0.05) induction/inhibition is indicated (*). Figure 5: Effects of Sp1/Sp4 downregulation and the ChIP assay on the G9a and \(\beta\)1-integrin promoters in (**A**).