8 research outputs found

    Analysis of the protein expression of tight junctions and cytoskeleton—IPEC-1.

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    <p>Different proteins of the tight junctions and cytoskeleton were analysed by Western blot and immunofluorescence: CK18, β-actin, ZO-1, occludin, claudin-3 and claudin-4. At day 2 of culture IPEC-1 showed in the Western blots a weak protein expression of all proteins studied. A strong ZO-1 and occludin immunoreactivity was found at the border of the cells. In IPEC-1 ZO-1 showed a low expression, whereas a large amount of occludin was present. The cytoskeleton proteins CK18 and actin were strongly expressed at the border of the IPEC-1 cells. Furthermore, no stress fibres were detected in the cells. Claudin-3 and claudin-4 were observed in the cells. A spot like character of claudin-3 distribution in the area where at least three cells were closely located. Claudin-4 showed an expression at the cell border but also within the cytoplasm. (blue = DAPI; bar = 20 μm)</p

    Polarisation of IPEC-1 and IPEC-J2, confocal microscopy and transmission electron microscopy.

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    <p>(A) Ten days old IPEC-1 and IPEC-J2 cells were fixed and stained for ZO-1 (<i>Zonula occludens</i>, green) and beta-catenin (<i>Zonula adherens</i>, red). Both cells lines showed a polarised structure with ZO-1 immunoreactivity at the apical pole and the underlying beta-catenin expression. (B) Important genes of a polarised brush border were analysed using microarray and qPCR. Here, significant differences were found between the cell lines. VIL1 (villin-1), VIL2 (villin-2 = ezrin), TLR4 (toll like receptor 4), MUC4 (mucin 4) and ESPN (espin) were significant up-regulated in IPEC-J2 in comparison to IPEC-1 in microarray as well as in qPCR. Villin1 and villin2 (= ezrin) were followed up by Western blot analyses. No villin-1 was detected in IPEC-1 but a strong expression in IPEC-J2 over time. Villin-2 was expressed in both cell lines. (C) Cells were cultured for 10 or 31 days and analysed using transmission electronmicroscopy. Both cell lines showed well-developed tight junctions at day 10 and 31 (red arrows). Desmosomes (blue arrows) and interdigitation (yellow arrow) were also observed. No differences were detected concerning microvilli length between both cell lines and at both time points. In contrast, the number of microvilli differed between the cell lines (IPEC-J2>IPEC-1).</p

    Analyses of important genes of the metabolism and oxygen-consumption.

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    <p>Important genes of the metabolism were analysed in both cell lines cultured on membranes for 10 days using microarray analyses and qPCR (A). PDHB (pyruvate dehydrognase subunit B) and CYC1 (cyctochrome C) are significantly down-regulated in the microarray and qPCR. SDH (succinate dehydrogenase subunit B) and HIF1a (hypoxia inducible factor 1a) are both significantly up-regulated in the microarray but not in qPCR. Furthermore, oxygen-consumption of both cell lines cultured on dishes or membranes for 10 days was examined. Both cell lines showed a significant higher O<sub>2</sub>-consumption on membranes (IPEC-1: 20.07 nmol/100 000 cells; IPEC-J2: 75.27 nmol/100 000 cells) in comparison to dishes (IPEC-1: 3.18 nmol/100 000 cells; IPEC-J2: 8.18 nmol/100 000 cells). At the same time, a significant higher oxygen-consumption was found in IPEC-J2 in comparison to IPEC1, which was independent of the culture support.</p

    Anchorage independent growth.

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    <p>Both cell lines were seeded in “Soft agar” (S, 0.33%) on “Feeder agar” (F, 0.5%) with (w) or without (wo) the application of EGF and ITS. Caco-2 cells were used as positive control. No anchorage independent growth was detected in IPEC-1 and IPEC-J2. On the other hand, Caco-2 showed an anchorage independent growth which did not depend on the additives EGF and ITS (bar = 20 μm). The results represent at least three independent experiments (n = 3).</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