Polyphosphate Maintains Intestinal Homeostasis Figure 5. Poly P prevented [3H]-mannitol flux induced by exposure to an oxidant, and integrin and p38 antagonist inhibited its protective action. (A) 10 or 100 mg/mL of poly P inhibited transmural [3H]-mannitol fluxes in the presence or absence of 0.3 mM NH2Cl (n = 5, each group). (B–F) Inhibition of 0.3 mM NH2Cl-induced transmural [3H]-mannitol fluxes by 100 mg/mL of poly P was abolished by 1 mM of p38 MAPK inhibitor SB203580 (B), peptide antagonist of integrin, 1 mM of echistatin or 10 mM of Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP) (C), 10 mM of reverse sequence of GRGDS, Ser-Thr-Asp-Gly-Arg-Gly (D), 1 mg/mL of human integrin a3b1 (E), 1 mM of cytochalasin D (F), 1 mM of EGFR inhibitor AG1478 or 1 mM of PDGFR inhibitor AG1296 treatment (n = 5, each group). *, ** Significantly different from corresponding control group at P,0.05 and P,0.01, respectively. doi:10.1371/journal.pone.0023278.g005 Isolation of HSP27-inducible substance from L. brevis SBC8803 culture supernatant The culture medium was centrifuged at 5006 g for 10 min to obtain the culture supernatant, which was then filtered through a 0.2-mm membrane. While stirring, ammonium sulfate was added to the supernatant to give 65% saturation, and then the mixture was centrifuged at 50006 g for 10 min. Further, ammonium sulfate was added to the obtained supernatant to give 90% saturation, and then the mixture was centrifuged at 50006 g for 10 min. The precipitates were redissolved in distilled water, and then desalted using a dialysis tube equipped with molecular weight 7000 cutoff membrane (SnakeSkin dialysis tube, Thermo Scien- tific, Rockford, IL). The ammonium sulfate precipitate was separated by anion-exchange chromatography. The sample was applied to a DEAE Sephadex A-50 column (GE Healthcare, Buckinghamshire, UK). The column was eluted with 0 M, 0.1 M, 0.5 M, and 1.0 M NaCl dissolved in 20 mM Tris-HCl (pH 8.5) stepwise, and each fraction was collected. Next the HSP27- inducible fraction obtained from the DEAE anion-exchange chromatography was separated by size-exclusion chromatography. The sample was applied to a Sepahdex G-100 (GE Healthcare), and eluted with 20 mM phosphate buffer (pH 6.5). Finally, the sample was separated using an A¨KTA design HPLC system (GE Healthcare) using a Shodex KW800 column (300 mm68 mm ID, Showa denko, Tokyo, Japan). The sample solution was eluted with 20 mM phosphate buffer (pH 6.5) at a flow rate of 0.1 ml/min. The eluent was monitored with an ultraviolet spectrophotometer at 220 nm. Quantification of poly P Polyphosphate was quantified by metachromatic assay with the TBO method, and phosphoric acid was quantified by the Mo-blue method after acid hydrolysis of Poly P. The TBO method was based on the decrease in absorbance at 620 nm by the metachromatic reaction of TBO with poly P. Ten microliters of sample solution was mixed with 500 mL of TBO assay solution (15 mg/mL TBO and 0.1 N acetic acid) and left for 15 min at room temperature. The absorbance at 620 nm was measured within 30 min. For the Mo-blue method, 5 mL of sample solution was mixed with an equal volume of 2 N HCl and heated at 95uC for 30 min for acid degradation of poly P. The solution was then diluted to 300 mL with distilled water, mixed with 700 mL of Mo-blue assay solution (0.42% (NH4)6Mo7O24 4H2O dissolved in 1 N H2SO4:10% ascorbic acid = 6:1), and incubated at 45uC for 20 min. Phosphoric acid concentration was determined from the absorbance at 750 nm and calculated from a standard phosphoric acid solution curve. Synthesis and degradation of poly P by PPK To synthesize poly P, 1 mL of reaction mixture containing 50 mM Tris-HCl (pH 7.4), 40 mM ammonium sulfate, 4 mM MgCl2, 40 mM creatine phosphate, 20 ng/mL creatine kinase, 1 mM ATP (pH 7.2), and 1 U of polyphosphate kinase (PPK) from Propionibacterium shermanii was incubated for 3 h at 37uC. To purify enzymatically synthesized poly P from the reaction mixture, 100 mL of 50 mM CaCl2 was added to the reaction mixture to aggregate the synthesized poly P and then centrifuged at 50006 g for 10 min. The precipitates were dissolved by adding 50 mM ethylenediaminetetraacetic acid (EDTA) solution, and then low- molecular weight components such as Ca2+ ions and EDTA were removed from the solution by dialysis using a tube equipped with 3-kDa MWCO membrane. To degrade poly P, 2 mL of poly P solution was incubated at 37uC for 3 h in the final reaction mixture (50 mmol/L (NH4)2SO4, 4 mmol/L MgCl2, 10 mmol/L ADP, 40 mmol/L HEPES-KOH [pH 7.5], and 1 U/mL PPK). Western blots After washing Caco2/BBE cells with PBS, proteins were extracted from samples using a Mammalian Cell Extraction Kit (BioVision, Mountain View, CA) and analyzed by Western blotting. Ten to thirty micrograms proteins of each sample was resolved by SDS-PAGE (12.5%) and immediately transferred to a nitrocellulose membrane using a transfer buffer (25 mM Tris, pH 8.8, 192 mM glycine with 20% (vol/vol) methanol). Nitrocel- lulose membranes were incubated in PBS with 0.05% (vol/vol) Tween 20 (T-PBS) containing 5% (wt/vol) skim milk or 1% (wt/ vol) bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) for 1 h at room temperature to block nonspecific binding. The blot was incubated overnight at 4uC with HSP27, HSC70 antibody (Stressgen, Victoria, British Columbia, Canada), anti-human p38 MAPK and phospho-p38 MAPK, anti-human extracellular signal- regulated kinase (ERK) and phospho-ERK, anti-human c-Jun N- terminal kinase (JNK), phospho-JNK, anti-human phosphatidyli- nositol 3-kinase (Akt), phospho-Akt, anti-human NF-kB p65, phospho-NF-kB p65, IkB (Cell Signaling Technology, Danvers, MA) or anti-human b-actin (Imgenex, San Diego, CA) as the primary antibody. Blot was washed 3 times for 10 min each in T- PBS at room temperature, incubated for 60 min in species- appropriate horseradish peroxidase-conjugated secondary anti- body (R&D systems, Minneapolis, MN) in T-PBS, washed 3 times in T-PBS, and developed using the Super-Signal West Pico enhanced chemiluminescence system (Pierce Chemical, Rockford, IL). Real-time RT-PCR Total RNA was extracted from frozen mouse colon using Trizol (Invitrogen), and purified with an RNeasy mini kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. The quality and quantity of total RNA was verified by agarose gel electrophoresis and spectrophotometry. Genomic DNA was eliminated, and single-stranded complementary DNA (cDNA) was synthesized using a QuantiTect reverse transcription kit (Qiagen). Gene expression was measured by real-time RT-PCR using a LightCycler 480 (Roche Applied Science) using specific primers (sense, 59-tccaggatgaggacatgagcac-39, anti-sense, 59-gaa- cgtcacacaccagcaggtta-39 for the analysis of mouse IL-1b, sense, 59- aagccagagctgtgcagatgagta-39, anti-sense, 59-tgtcctgcagccactggttc-39 for the analysis of mouse IL-6, sense, 59-gaccagctggacaacatactgc- PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23278