Ll lysates was isolated and DNAse digestion was performed as recommended by the supplier to avoid genomic DNA contamination (RNeasy Mini Kit, Qiagen, Hilden, Germany). Subsequently 1 mg of total RNA was reverse transcribed into cDNA with oligo (dT) primers and 15 U/mg AMV Reverse Transcriptase (Promega, Madison, USA) according to standard procedures. RNA preparations were used for PCR analysis.Quantitative Real-time Reverse Transcriptase PCRFor mRNA quantification, real-time PCR was performed in a SYBR Green fluorescence temperature cycler (LightCyclerH, Roche Diagnostics, Mannheim, Germany). Single-stranded cDNA (or gene-specific plasmids as controls) corresponding to 10 ng of RNA served as a template for PCR with specific oligonucleotide primer pairs (table 2) as described previously [31]. All primers were checked for specific binding to the sequence of interest using BLAST. Plasmids for each product were synthesized with the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. PCR-amplified DNA fragments were confirmed by sequencing. The correctly sequenced plasmids were serially diluted for internal standard curves. The mRNA data were normalized to the mRNA of ?actin.Mouse TissueColonic samples (mRNA and 223488-57-1 biological activity tissue from whole mouse colonic mucosa) of mice housed in AKT inhibitor 2 site germfree conditions at the University of Ulm, mice housed in specific pathogen free (SPF) conditions at the University of Cologne and germfree mice that were transferred to the SPF facility in Cologne for cohousing with SPF mice for 4 weeks (conventionalized) (all C57Bl/6), were kindly provided byProtein PreparationLS174T cells were washed with PBS, harvested by scraping and centrifuged for two times at 1300 rpm for 5 minutes. Whole cell lysates were extracted with lysis buffer containing 20 mM TrisBacteria Regulate Intestinal DifferentiationFigure 3. HBD2, Muc1 and Muc2 mRNA expression in LS174T cells after treatment with different heat-inactivated bacteria for 3 hours. HBD2 expression was induced by Symbioflor G2, E. coli K-12, E. coli Nissle 1917, B. breve and adolescentis (A). Muc1 transcripts were upregulated by Symbioflor G2, E. coli K-12, E. coli Nissle 1917, L. fermentum and acidophilus as well as B. breve (B). Muc2 mRNA was unchanged (C). Data represent the means 6 SEM normalised to basal expression of untreated controls set at 1 (n = 4). *: p,0.05, **: p,0.01, ***: p,0.001. For 12 hours treatment results see Fig. S2. doi:10.1371/journal.pone.0055620.gHCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 Trition X-100, 25 mM Sodiumpyrophosphat, 1 mM Glycerolphosphat, 1 mM Na3VO4, 6M Urea and 1 Protease Inhibitor Cocktail (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Mouse colonic tissue was extracted with lysis buffer (see above) and homogenized by FastPrep instrument. Mouse and cell culture lysates were centrifuged at 13000 rpm at 4uC for 20 minutes and the supernatant was collected. Total protein amount was measured with the Bicinchoninic Acid Protein Assay (Smith) as described previously [32]. Isolated proteins were used for Western blot experiments.Western Blot40 mg (LS174T cell lysates) or 20 mg (mouse colonic tissue) of total protein was separated on a 10 Tris-glycin SDS polyacrylamide gel, transferred to 0.45 mm pore size nitrocellulose membranes (Schleicher Schuell, Keene, NH, USA) and blockedwith 5 skimmed milk powder in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.05 Tween 20) for 1 hour. Then, the membranes were washed with TBST a.Ll lysates was isolated and DNAse digestion was performed as recommended by the supplier to avoid genomic DNA contamination (RNeasy Mini Kit, Qiagen, Hilden, Germany). Subsequently 1 mg of total RNA was reverse transcribed into cDNA with oligo (dT) primers and 15 U/mg AMV Reverse Transcriptase (Promega, Madison, USA) according to standard procedures. RNA preparations were used for PCR analysis.Quantitative Real-time Reverse Transcriptase PCRFor mRNA quantification, real-time PCR was performed in a SYBR Green fluorescence temperature cycler (LightCyclerH, Roche Diagnostics, Mannheim, Germany). Single-stranded cDNA (or gene-specific plasmids as controls) corresponding to 10 ng of RNA served as a template for PCR with specific oligonucleotide primer pairs (table 2) as described previously [31]. All primers were checked for specific binding to the sequence of interest using BLAST. Plasmids for each product were synthesized with the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. PCR-amplified DNA fragments were confirmed by sequencing. The correctly sequenced plasmids were serially diluted for internal standard curves. The mRNA data were normalized to the mRNA of ?actin.Mouse TissueColonic samples (mRNA and tissue from whole mouse colonic mucosa) of mice housed in germfree conditions at the University of Ulm, mice housed in specific pathogen free (SPF) conditions at the University of Cologne and germfree mice that were transferred to the SPF facility in Cologne for cohousing with SPF mice for 4 weeks (conventionalized) (all C57Bl/6), were kindly provided byProtein PreparationLS174T cells were washed with PBS, harvested by scraping and centrifuged for two times at 1300 rpm for 5 minutes. Whole cell lysates were extracted with lysis buffer containing 20 mM TrisBacteria Regulate Intestinal DifferentiationFigure 3. HBD2, Muc1 and Muc2 mRNA expression in LS174T cells after treatment with different heat-inactivated bacteria for 3 hours. HBD2 expression was induced by Symbioflor G2, E. coli K-12, E. coli Nissle 1917, B. breve and adolescentis (A). Muc1 transcripts were upregulated by Symbioflor G2, E. coli K-12, E. coli Nissle 1917, L. fermentum and acidophilus as well as B. breve (B). Muc2 mRNA was unchanged (C). Data represent the means 6 SEM normalised to basal expression of untreated controls set at 1 (n = 4). *: p,0.05, **: p,0.01, ***: p,0.001. For 12 hours treatment results see Fig. S2. doi:10.1371/journal.pone.0055620.gHCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 Trition X-100, 25 mM Sodiumpyrophosphat, 1 mM Glycerolphosphat, 1 mM Na3VO4, 6M Urea and 1 Protease Inhibitor Cocktail (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Mouse colonic tissue was extracted with lysis buffer (see above) and homogenized by FastPrep instrument. Mouse and cell culture lysates were centrifuged at 13000 rpm at 4uC for 20 minutes and the supernatant was collected. Total protein amount was measured with the Bicinchoninic Acid Protein Assay (Smith) as described previously [32]. Isolated proteins were used for Western blot experiments.Western Blot40 mg (LS174T cell lysates) or 20 mg (mouse colonic tissue) of total protein was separated on a 10 Tris-glycin SDS polyacrylamide gel, transferred to 0.45 mm pore size nitrocellulose membranes (Schleicher Schuell, Keene, NH, USA) and blockedwith 5 skimmed milk powder in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.05 Tween 20) for 1 hour. Then, the membranes were washed with TBST a.
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