*2.3. NF-*κ*B Reporter Gene Assay*

DON, NX-3, AURO and their combinations were prepared in reaction tubes to be further diluted 1:100 by the addition of the THP1-Lucia™ cell suspension. Cells were counted, centrifuged for 5 min at 250 × *g* and resuspended at 1 × 10<sup>6</sup> cells/mL in fresh, prewarmed growth medium. Cell suspension was added to the respective toxin preparations, mixed gently and transferred into a 96-well plate (100 μL/well). After 2 h of incubation at 37 ◦C and 5% CO2, cells were treated with LPS (10 ng/mL) and incubated for an additional 18 h. 1% (*v*/*v*) DMSO with and without LPS treatment were used as solvent control. Heat-killed *Listeria monocytogenes* (HKLM; 20 × 10<sup>6</sup> cells/well) were used as positive control for Toll-like receptor-mediated activation of the NF-κB pathway. Following treatment, cells were centrifuged (250 × *g*, 5 min) and 10 μL of each supernatant were collected and the reporter gene assay was performed according to the manufacturer's protocol. For luminescence measurements QUANTI-Luc™ (Invivogen, San Diego, CA, USA), containing the luciferase substrate coelenterazine, was used.

In parallel cellular metabolic activity was monitored by the alamarBlue® assay (Invitrogen, Carlsbad, CA, USA). After supernatant collection for luciferase activity measurements, 10 μL of alamarBlue® reagen<sup>t</sup> (Invitrogen, Carlsbad, CA, USA) were added to the cells and incubated for 2 h. Subsequently, 50 μL/well were transferred to a black 96-well plate and fluorescence intensity was measured at 530/560 nm (excitation/emission). Both luciferase activity and fluorescence intensity measurements were performed on a SynergyTM H1 hybrid multimode reader (BioTek, Bad Friedrichshall, Germany), assessing at least five independent experiments in duplicates.

### *2.4. Quantitative Analysis of Cytokine Gene Transcription*

Gene transcription levels of up to four pro-inflammatory cytokines in two colon cell lines (HT-29: TNF-<sup>α</sup>, IL-1β, IL-8; HCEC-1CT: TNF-<sup>α</sup>, IL-1β, IL-8, IL-6) were analyzed by quantitative real-time PCR (qRT-PCR). Cells were seeded in 12-well plates (HT-29: 150,000 cells/well; HCEC-1CT: 50,000 cells/well) and allowed to grow for 48 h. Cells were incubated for 5 h, consisting of 2 h preincubation with the test compounds (DON, NX-3, AURO and the respective combinations) followed by IL-1β co-treatment (25 ng/mL) for additional 3 h. Total RNA was extracted using Maxwell® 16 Cell LEV Total RNA Purification Kits (Promega, Madison, WI, USA) and reversed transcribed into complementary DNA (cDNA) by QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. cDNA samples were amplified in duplicates in presence of gene specific primers (QuantiTect® Primer Assays, Qiagen, Hilden, Germany) and QuantiTect® SYBR Green Master Mix (Qiagen, Hilden, Germany) using a StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA). The following primer assays were used: β-actin (ACTB1, Hs\_ACTB1\_1\_SG, QT00095431); glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs\_GAPDH\_1\_SG, QT00079247); TNF-α (Hs\_TNF\_1\_SG; QT00029162); IL-1β (Hs\_IL1B\_1\_SG, QT00021385); IL-8 (Hs\_CXCL8\_1\_SG; QT00000322); IL-6 (Hs\_IL6\_1\_SG, QT00083720). The applied PCR protocol included 15 min enzyme activation at 95 ◦C, 45 cycles of 15 s at 94 ◦C, 30 s at 55 ◦C and 30 s at 72 ◦C. For the quantification of the fluorescence signal and further data analysis, StepOnePlus® software (Applied Biosystems, Foster City, CA, USA) was used. For each tested sample, at least five independent experiments were performed. Presented transcript data were normalized to the mean of transcript levels of endogenous control genes (ACTB1, GAPDH) by applying the ΔΔCt-method [35] for relative quantification. In relation to the unchallenged solvent control, IL-1β-stimulationenhanced cytokine transcription levels of TNF-<sup>α</sup>, IL-1β, IL-8 and IL-6 already 4200-, 500-, 4700- and 1600-fold, respectively.

### *2.5. Determination of Cellular Protein Content and Metabolic Activity*

To determine effects on the cellular protein content, the metabolic viability, and to preclude cytotoxic effects for qRT-PCR experiments, the sulforhodamine B (SRB) assay according to Skehan, et al. [36] and the alamarBlue® assay were performed in HT-29 and HCEC-1CT cells. HT-29 (5500 cells/well) and HCEC-1CT cells (2000 cells/well) were seeded into 96-well plates and allowed to grow for 48 h. Cells were incubated for 5 h, including preincubation for 2 h with the substances (DON, NX-3, AURO and the respective combinations) followed by 3 h IL-1β co-treatment (25 ng/mL). Following 4 h of incubation, 10 μL alamarBlue® reagen<sup>t</sup> were added to the incubation solution. After 75 min, 70 μL of the supernatant were transferred into a black 96-well plate and fluorescence intensity was measured at 530/560 nm (excitation/emission) using a SynergyTM H1 hybrid multimode reader (BioTek, Bad Friedrichshall, Germany). Subsequent to the fluorescence readout, cells were rinsed once with prewarmed PBS, fixed by the addition of 5% (*v*/*v*) trichloroacetic acid and incubated at 4 ◦C for 30 min. After the fixation, plates were washed four times with water, dried overnight at room temperature and stained for 1 h by adding a solution of 0.4% (*w*/*v*) SRB in 1% (*v*/*v*) acetic acid. Plates were washed twice with water and 1% acetic acid solution and dried at room temperature in the dark. Finally, 10 mM Tris buffer (pH 10; 100 μL/well) was added to dissolve the dye, and single wavelength absorbance (570 nm) was measured using a SynergyTM H1 hybrid multimode reader (BioTek, Bad Friedrichshall, Germany). One percent (*v*/*v*) water (LC–MS grade) and 1% (*v*/*v*) DMSO with and without IL-1β stimulation served as solvent control, whereas 1% (*v*/*v*) triton X-100 was used as positive control. Cell-free blank values were subtracted and measured data were referred to the respective solvent control. Each cell line was tested in duplicate with a minimum of five independent experiments.
