32 research outputs found

    Effect of glucose and lactate on cellular ATP content in IPEC-1 cells after 1 h treatment.

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    <p>IPEC-1 cells were grown to confluence (7 d) and treated for 1 h with HBSS-based solutions. Supernatant was removed and ATP content was measured by a luminescence based assay. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

    Expression of selected genes of cellular processes by microarray and qPCR in membrane cultured IPEC-J2 cells in response to apical and basolateral DON application.

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    <p>Regulation of transcripts of (<b>A</b>) LAMP2 (lysosomes), (<b>B</b>) CDKN1A (cell cycle), (<b>C</b>) CTNNB1 (focal adhesion) and (<b>D</b>) CLDN3 (tight junction) in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. The mRNA levels are given as fold increase over untreated control. Bars represent means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post test (* p≤0.05; ** p≤0.01).</p

    Expression of selected metabolic genes measured by microarray and qPCR in membrane cultured IPEC-J2 cells in response to apical and basolateral DON application.

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    <p>Regulation of transcripts of (<b>A</b>) PDHA1, (<b>B</b>) SDHB and (<b>C</b>) CYC1 in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. mRNA levels are given as fold increase over untreated control. Bars represent means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post hoc test (* p≤0.05; ** p≤0.01).</p

    Analysis of cell cycle specific genes on mRNA and protein level.

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    <p>The expression of p53 was analysed by microarray, qPCR and Western blot. (A) IPEC-J2 showed a 27.4 fold increase of the p53-mRNA level in comparison to IPEC-1. This was confirmed by qPCR and a 300-fold increase was detected in IPEC-J2. On the other hand, no protein expression was observed in IPEC-1/IPEC-J2 and Caco-2 as well as in the cells, which were treated with the p53-activator. In the next step, the expression of BAX, BAD and BCL-X was analysed using microarray, qPCR and Western blot. (B) BAD showed a 1.8 fold increase in IPEC-J2 in the microarray. Same results were found in qPCR, but no differences in the protein expression were found between the cell lines. A significant BCL-X up-regulation was detected in the microarray in IPEC-J2 but no differences were present between IPEC-1 and IPEC-J2 in the Western blot analyses. No BAX-protein was observed in both cell lines. When the total RNA and protein content was examined, significant differences were found between the cell lines. IPEC-J2 showed a higher RNA- and protein content in comparison to IPEC-1. RPL10A as important gene of the 60S ribosomal subunit was significantly decreased in the microarray but not in qPCR in IPEC-J2 in comparison to IPEC-1. Western blot analyses showed a slightly decrease of the protein in IPEC-J2 over time. (C) The RNA and protein content per cell was measured in both cell lines. IPEC-J2 showed a significant higher level in RNA and proteins. On the other hand, a slight decrease on protein level of RPL10A was found in IPEC-J2 in comparison to IPEC-1 in Western blot analyses.</p

    Comparison of microarray data and qPCR.

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    <p>*p≤0.05;</p><p>**p≤0.01;</p><p>***p≤0.001</p><p>Important genes of the significantly regulated pathways shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132323#pone.0132323.t002" target="_blank">Table 2</a> were analysed via qPCR. The microarry data are shown and two reference genes were used to illustrate significant differences between both cell lines. Asterisks indicate significant differences from IPEC-1.</p

    Deoxynivalenol (DON) enlarged nucleic area (µm<sup>2</sup>) of IPEC-J2 cells.

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    <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

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

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    <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

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

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    <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

    Quantification of cytosol / nucleus NADH ratio in IPEC-1 cells after 1 h treatment with glucose and lactate without further challenge.

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    <p>Cellular autofluorescence of IPEC-1 cells was monitored at ex: 340 nm / em: 510 nm. <b>(A)</b> Change in spatial distribution of autofluorescence in response to Anti A (100 ÎĽM). <b>(B)</b> Cytosolic autofluorescence in response to complex III inhibitor Anti A (100 ÎĽM) and uncoupler FCCP (1 ÎĽM). <b>(C)</b> IPEC-1 cells were treated as indicated for 1 h. Glucose (gluc) concentration was 1 g/L if not otherwise indicated. Cells were treated with lactate (lac) 25 mM or increased gluc 4.5 g/L. Autofluorescence measured after 1 h and ratio of cytosolic (F<sub>cytosol</sub>) and nuclei (F<sub>nucleus</sub>) was calculated (ex: 340 nm / em: 510 nm) from individual cells without further stimulation. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p
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