Effect of deoxynivalenol (DON) on total cell count of IPEC-J2.

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. Data are given as means (±SEM) in triplicates from three separate experiments. ***p≤0.001 vs. DON0.</p

Western blot of tight junction proteins ZO-1 and claudin-3 in IPEC-J2 cells treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0 or 2000 ng/mL) applied from apical or basolateral side in complete medium. ZO-1 (225 kDa) and claudin-3 (22 kDa) expression was analysed by immunoblotting. The housekeeping protein GAPDH (37 kDa) was used as loading control.</p

Cellular distribution of the tight junction protein claudin-3 (CLDN-3) in IPEC-J2 monolayers treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein claudin-3 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.</p

Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.</p

Deoxynivalenol (DON) enlarged nucleic area (µm²) of IPEC-J2 cells.

<p>xCells were incubated for 24, 48 or 74 hours with DON (0–4000 ng/mL) in complete medium applied from apical (ap) or basolateral (bl) side. Data are given as means (±SEM) from three separate experiments.</p><p>***p≤0.001 vs control.</p

Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.

<p>Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm²) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.</p

Cell cycle analysis of IPEC-J2 cells treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and synchronised for 24 hours in serum free medium and then incubated for 24, 48 or 74 hours with DON (0 or 2000 ng/mL) applied from apical or basolateral side in complete medium. After staining with propidium iodide DNA content was analysed by FACS. Data given are means (±SEM) from five separate experiments. **p≤0.01 vs. DON0, ***p≤0.001 vs. DON0.</p

Effect of deoxynivalenol (DON) on apoptosis of IPEC-J2 cells.

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0, 200 or 2000 ng/mL) applied from apical or basolateral side in complete medium. Protein expression of pro caspase 3 (35 kDa) cleaved caspase 3 (17 kDa) was analysed by immunoblotting. The housekeeping protein GAPDH (37 kDa) was used as loading control. Staurosporine (100 µM) was used as positive control for cleaved caspase 3.</p

Generally regulated genes found in all treatments investigated independent of applied DON concentration and application route.

<p>Generally regulated genes found in all treatments investigated independent of applied DON concentration and application route.</p

Number of regulated IPEC-J2 genes after 72 h apical and basolateral DON treatment analysed by microarray.

<p>Membrane cultures were treated with 200 and 2000 ng/mL DON from apical and basolateral side for 72 h. Isolated mRNA was analysed with GeneChip® Porcine Genome Array. Number of significantly (p<0.05) up- or down-regulated genes in comparison to untreated control genes are given. Values represent numbers of annotated and non-annotated genes of three independent experiments.</p