Colon cancer cells and macrophage cells After demonstrating that systemic treatment with EKODE increased colitis and tumor inflammation in vivo, we tested irrespective of whether EKODE straight acted on colon cancer cells or immune cells to induce inflammation. To perform so, we treated colon cancer (HCT-116) cells or macrophage (RAW 264.7) cells with 300 nM EKODE, then examinedinflammatory responses. We’ve determined the concentration of 300 nM, since this can be similar to the concentrations of endogenous EKODE in the colon of AOM/DSS-induced CRC mice (Fig. 2D). In HCT-116 cells, treatment with EKODE induced gene expression of pro-inflammatory cytokines (IL-6, IFN-, TNF-) right after 24-h treatment, demonstrating its potent pro-inflammatory impact (Fig. 7A). Next, we tested the effect of EKODE on NF-B, which is an essential signaling pathway involved in inflammation [14]. Following 300 min treatment,L. Lei et al.Redox S1PR5 Agonist drug Biology 42 (2021)Fig. 5. EKODE induces intestinal barrier dysfunction and increases LPS/bacterial translocation. A, LPS concentration in plasma (n = six mice per group). B, Gene expression of 16S rRNA gene in blood and spleen (n = 4 mice per group). C, Gene expression of Il-1, Tnf- and Il-10 in spleen (n = 4 mice per group). D, Gene expression of Occludin in colon (n = five mice per group). E, IHC staining of Occludin in colon (n = six mice per group, scale bars: 50 m). The results are imply SEM. The STAT3 Inhibitor Formulation statistical significance of two groups was determined applying Student’s t-test or Wilcoxon-Mann-Whitney test.Fig. 6. Remedy with EKODE exaggerates AOM/DSS-induced colon tumorigenesis in mice. A, Scheme of animal experiment (dose of EKODE = 1 mg/kg/day). B, Quantification of colon tumor in mice (n = 8 mice per group). C, H E histology and IHC staining of PCNA and -catenin in colon (n = eight mice per group, scale bars: 50 m). D, Gene expression of Mcp-1, Il-6, Ifn-, Pcna, Myc, Jun, Ccnd-1 and Vegf in colon (n = eight mice per group). The results are expressed as indicates SEM. The statistical significance of two groups was determined utilizing Student’s t-test or Wilcoxon-Mann-Whitney test.L. Lei et al.Redox Biology 42 (2021)Fig. 7. EKODE induces inflammation in human colon cancer HCT-116 cells and mouse macrophage RAW 264.7 cells. The cells were treated with 300 nM EKODE or car (DMSO). A, EKODE elevated gene expression of pro-inflammatory cytokines in HCT-116 cells immediately after 24-h treatment (n = five per group). B, EKODE enhanced IB degradation in HCT-116 cells (n = three per group). C, EKODE improved nuclear translocation of p65 in HCT-116 cells (n = three per group). D, EKODE increased gene expression of pro-inflammatory cytokines in RAW 264.7 cells following 24-h remedy (n = five per group). B, EKODE enhanced IB degradation in RAW 264.7 cells (n = three per group). C, EKODE increased nuclear translocation of p65 in RAW 264.7 cells (n = three per group). The outcomes are imply SEM. The statistical significance of two groups was determined utilizing Student’s t-test or Wilcoxon-Mann-Whitney test. The cell culture experiments had been performed with no less than three independent repeats.EKODE induced degradation of IB- and enhanced nuclear translocation of p65, demonstrating that it activated the NF-B signaling pathway (Fig. 7B ). A equivalent result was also observed in RAW 264.7 cells (Fig. 7D ). General, these outcomes demonstrate that therapy with EKODE, at nM doses, induced inflammatory responses and activated NF-kB pathway in each colon cancer cells and macrophage cells, illustrating its potent pro-inflammatory e.