F the TA-01 control (Fig. 3). For Cry1Ab treatment an increase of the delta cell index was observed buy (��)-Hexaconazole during the first hours after treatment. Thereafter it returns to theFigure 1. Effect of Cry1Ab on viability of IPEC-J2 cells. IPEC-J2 cells were incubated with indicated concentrations of Cry1Ab for 24 h and 48 h, respectively. Data are from one representative experiment out of 2 performed. (A) The Cell Proliferation Reagent WST-1 (Roche) was used to measure mitochondrial dehydrogenase activity of viable cells. Valinomycin was used as cell damaging control. Bars represent the mean +/2S.D. of 5 replicate wells. (B) The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to estimate the number of viable metabolically active cells based on quantitation of ATP. Bars represent the mean +/2S.D. of 5 replicate wells. (C) The CytoTox-ONETM Homogenous Membrane Integrity Assay (Promega) was used for estimating the number of 16574785 dead cells by release of LDH from damaged cells. Bars represent the mean +/2S.D. of 12 replicate wells. doi:10.1371/journal.pone.0067079.gImpact of Cry1Ab on Porcine Intestinal CellsFigure 2. Effect of Cry1Ab on transepithelial electrical resistance (TEER) in porcine intestinal epithelial monolayers. TEER of IPEC-J2 cell monolayers was measured 24 h and 48 h after application of increasing doses of Cry1Ab (0.1, 0.5, 1 mg/ml) or valinomycin (500 nM) at the apical side [means 6 standard deviation; of initial value t = 0 h]; n = 3 independent experiments (2? cell culture inserts per treatment group per experiment). doi:10.1371/journal.pone.0067079.gbaseline of the control cells (after 24 h) indicating a mild short term and reversible effect of Cry1Ab.Protein Expression Changes in Response to Cry1AbTo analyze global protein expression changes induced by Cry1Ab a quantitative proteome analysis of porcine intestinal cells (IPEC-J2) was performed. After 24 h incubation with a nonphysiological high concentration of Cry1Ab (1 mg/ml) proteinFigure 3. Dynamic monitoring of IPEC-J2 cell response after treatment with Cry1Ab and valinomycin using the xCELLigence system. IPEC-J2 cells of 10,000 cells/well in E-plate96 were observed during 24 h. Data points represent mean values +/2 SEM (n = 5 wells). doi:10.1371/journal.pone.0067079.gImpact of Cry1Ab on Porcine Intestinal Cellsextracts were prepared as described. Five biological replicates per treatment were reverse labelled by Cy3 and Cy5 and run together with the Cy2 labelled internal standard giving a total of 15 images. Using DeCyder Software analysis about 2500 spots were detected in each gel and quantified, normalized and inter-gel-matched.. Protein expression changes were considered as significant when their quantity decreased or increased by at least 1.3-fold (student’s T-test, p#0.05). Notably, Cry1Ab treatment caused only few modifications in protein expression profiles, only three proteins out of .2000 were differentially expressed (Table 1), one protein (heat-shock protein 70) was more abundant and two proteins (cytokeratin 8 and heterogeneous nuclear ribonucleoprotein) were less abundant. The location of these spots is indicated in Figure 4 and the 23977191 protein name, NCBI database accession number, Mascot score and statistics for the three identified proteins are given in Table 1. To validate the DIGE/MS results the selected up-regulated stress response biomarker Hsp70 was further quantified at the protein level. The induction of Hsp70 was determined by ELISA and verified by.F the control (Fig. 3). For Cry1Ab treatment an increase of the delta cell index was observed during the first hours after treatment. Thereafter it returns to theFigure 1. Effect of Cry1Ab on viability of IPEC-J2 cells. IPEC-J2 cells were incubated with indicated concentrations of Cry1Ab for 24 h and 48 h, respectively. Data are from one representative experiment out of 2 performed. (A) The Cell Proliferation Reagent WST-1 (Roche) was used to measure mitochondrial dehydrogenase activity of viable cells. Valinomycin was used as cell damaging control. Bars represent the mean +/2S.D. of 5 replicate wells. (B) The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to estimate the number of viable metabolically active cells based on quantitation of ATP. Bars represent the mean +/2S.D. of 5 replicate wells. (C) The CytoTox-ONETM Homogenous Membrane Integrity Assay (Promega) was used for estimating the number of 16574785 dead cells by release of LDH from damaged cells. Bars represent the mean +/2S.D. of 12 replicate wells. doi:10.1371/journal.pone.0067079.gImpact of Cry1Ab on Porcine Intestinal CellsFigure 2. Effect of Cry1Ab on transepithelial electrical resistance (TEER) in porcine intestinal epithelial monolayers. TEER of IPEC-J2 cell monolayers was measured 24 h and 48 h after application of increasing doses of Cry1Ab (0.1, 0.5, 1 mg/ml) or valinomycin (500 nM) at the apical side [means 6 standard deviation; of initial value t = 0 h]; n = 3 independent experiments (2? cell culture inserts per treatment group per experiment). doi:10.1371/journal.pone.0067079.gbaseline of the control cells (after 24 h) indicating a mild short term and reversible effect of Cry1Ab.Protein Expression Changes in Response to Cry1AbTo analyze global protein expression changes induced by Cry1Ab a quantitative proteome analysis of porcine intestinal cells (IPEC-J2) was performed. After 24 h incubation with a nonphysiological high concentration of Cry1Ab (1 mg/ml) proteinFigure 3. Dynamic monitoring of IPEC-J2 cell response after treatment with Cry1Ab and valinomycin using the xCELLigence system. IPEC-J2 cells of 10,000 cells/well in E-plate96 were observed during 24 h. Data points represent mean values +/2 SEM (n = 5 wells). doi:10.1371/journal.pone.0067079.gImpact of Cry1Ab on Porcine Intestinal Cellsextracts were prepared as described. Five biological replicates per treatment were reverse labelled by Cy3 and Cy5 and run together with the Cy2 labelled internal standard giving a total of 15 images. Using DeCyder Software analysis about 2500 spots were detected in each gel and quantified, normalized and inter-gel-matched.. Protein expression changes were considered as significant when their quantity decreased or increased by at least 1.3-fold (student’s T-test, p#0.05). Notably, Cry1Ab treatment caused only few modifications in protein expression profiles, only three proteins out of .2000 were differentially expressed (Table 1), one protein (heat-shock protein 70) was more abundant and two proteins (cytokeratin 8 and heterogeneous nuclear ribonucleoprotein) were less abundant. The location of these spots is indicated in Figure 4 and the 23977191 protein name, NCBI database accession number, Mascot score and statistics for the three identified proteins are given in Table 1. To validate the DIGE/MS results the selected up-regulated stress response biomarker Hsp70 was further quantified at the protein level. The induction of Hsp70 was determined by ELISA and verified by.
