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J Biol Chem 278: 36953C36958, 2003 [PubMed] [Google Scholar]. results were confirmed in infarcted myocardium where iNOS appearance was attenuated by Hsp90 inhibition in vivo markedly. Intriguingly, additional analyses demonstrated that inhibiting Hsp90 got no significant influence on the activation of either IKK-NF-B or JAK-STAT1 in LPS/IFN–stimulated cells. Neither was the nuclear transportation of dynamic STAT1 or NF-B suffering from Hsp90 inhibition. But Hsp90 inhibition markedly decreased the binding of active STAT1 and NF-B with their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was needed for STAT1 and NF-B bindings to iNOS promoters inside cells. These scholarly research disclose that besides performing as an allosteric enhancer, Hsp90 is necessary for transcriptional aspect binding amid iNOS mRNA transcription also. Because of the fundamental function of Hsp90 in iNOS gene transactivation, concentrating on Hsp90 might stand for a fresh method of intervene iNOS expression in diseases. for 15 min, as well as the supernatant was retrieved. Protein concentrations had been dependant on using the detergent-compatible proteins assay package (Bio-Rad). The proteins had been separated by SDS-PAGE, used in nitrocellulose membranes, and probed with the correct major antibodies. Membrane-bound major antibodies had been detected with supplementary antibodies conjugated with horseradish peroxidase. Immunoblots had been developed on movies using the improved chemiluminescence technique (SuperSignal Western world Pico, Pierce). RT-PCR. Total RNA of cultured cells of cardiac tissue had been extracted through the use of TRIzol Reagent (Invitrogen) based on the manufacturer’s guidelines. Change transcription was completed with the Great Capacity cDNA Change Transcription Package (Applied Biosystems). PCR was performed with Taq DNA polymerase. The next primers had been used for discovering iNOS: 5-GGGATGGCTTGCCCCTGG-3 and 5-CGGAGGCAGCACATCAAAG-3. Primers 5-CAAAGTTGTCATGGATGACC-3 and 5-GGTGAAGGTCGGAGTCAACG-3 were useful for measuring GAPDH. STAT1 and NF-B binding assays. The nuclei had been extracted from cells by initial incubating them in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4C for 15 min. Following the cells had been homogenized within a course douncer (15 strokes), cell homogenates had been spun at 3,000 for 5 min. The pellets had been retrieved, washed extensively, and resuspended in the nuclear removal buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, and 5 mM sodium pyrophosphate, protease inhibitors). The NF-B and STAT1 binding activity of nuclear ingredients had been measured using the TransFactor NF-B colorimetric package (Clontech, Mountain Watch) as well as the DuoSet mouse energetic STAT1 binding package (R&D Systems, Minneapolis), respectively, based on the manufacturer’s instructions. Chromatin immunoprecipitation. Organic 264.7 cells were treated with LPS (2 g/ml) or IFN- (100 U/ml) for 1 h in the existence and lack of geldanamycin. Formaldehyde (1%) was put into the culture moderate, and after incubation in the rocker for 10 min at area temperature, cells had been rinsed double with 4C ice-cold PBS and lysed for 10 min at 4C. After sonication, 20 l from the lysate had been utilized as DNA insight control. The rest of the lysate was diluted 10-fold with chromatin immunoprecipitation (ChIP) dilution buffer accompanied by incubation using the anti-NF-B p65 antibody (Santa-Cruz Biotechnology) or the anti-phospho-STAT1 (Tyr701) antibody (Cell Signaling Technology) over night at 4C. Immunoprecipitated complexes had been collected using proteins A/G Plus-agarose beads (Santa-Cruz Biotechnology). The precipitates had been extensively washed and incubated in the elution buffer (1% SDS and 0.1 M NaHCO3) at area temperature for 15 min. Cross-linking of protein-DNA complexes was reversed at 65C for 4 h. DNA was extracted using the Qiagen PCR purification package. ChIP assays handling NF-B used the PCR primers 5-CAAGCCAGGGTATGTGGTTT-3 (forward) and 5-GCAGCAGCCATCAGGTATTT-3 (reverse), resulting in a 290-bp fragment. ChIP assays for activated STAT1 binding to its IFN–regulated transcription factor STAT1 (GAS) site on the iNOS promoter used primers 5-ACACGAGGCTGAGCTGACTT-3 (forward) and 5-CACACATGGCATGGAATTTT-3 (reverse), resulting in a 186-bp fragment (24). The resulting product was separated by 2% agarose gel electrophoresis. Nitrite assay. Total nitrite released in cell culture medium was measured with a Griess reagent kit (Invitrogen). The reaction consisted of 20 l of Griess Reagent, 150 l of medium, and 130 l of deionized water. After incubation of the mixture for 30 min at room temperature, nitrite levels were measured at 548 nm using a M2 spectrophotometric microplate reader (Molecular Devices). In vivo myocardial infarction in mice. C57BCL/6 mice were purchased from Charles River Laboratories. Mice were maintained in a pathogen-free environment, and experiments.Hsp90 and the chaperoning of cancer. JAK-STAT1 in LPS/IFN–stimulated cells. Neither was the nuclear transport of active NF-B or STAT1 affected by Hsp90 inhibition. But Hsp90 inhibition markedly reduced the binding of active NF-B and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was essential for NF-B and STAT1 bindings to iNOS promoters inside cells. These studies reveal that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may represent a new approach to intervene iNOS expression in diseases. for 15 min, and the supernatant was recovered. Protein concentrations were determined by using the detergent-compatible protein assay kit (Bio-Rad). The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the appropriate primary antibodies. Membrane-bound primary antibodies were detected with secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique (SuperSignal West Pico, Pierce). RT-PCR. Total RNA of cultured cells of cardiac tissues were extracted by using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed with Taq DNA polymerase. The following primers were used for detecting iNOS: 5-GGGATGGCTTGCCCCTGG-3 and 5-CGGAGGCAGCACATCAAAG-3. Primers 5-GGTGAAGGTCGGAGTCAACG-3 and 5-CAAAGTTGTCATGGATGACC-3 were used for measuring GAPDH. NF-B and STAT1 binding assays. The nuclei were extracted from cells by first incubating them in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4C for 15 min. After the cells were homogenized in a class douncer (15 strokes), cell homogenates were spun at 3,000 for 5 min. The pellets were recovered, extensively washed, and resuspended in the nuclear extraction buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, and 5 mM sodium pyrophosphate, protease inhibitors). The NF-B and STAT1 binding activity of nuclear extracts were measured with the TransFactor NF-B colorimetric kit (Clontech, Mountain View) and the DuoSet mouse active STAT1 binding kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s instruction. Chromatin immunoprecipitation. RAW 264.7 cells were treated with LPS (2 g/ml) or IFN- (100 U/ml) for 1 h in the presence and absence of geldanamycin. Formaldehyde (1%) was added to the culture medium, and after incubation on the rocker for 10 min at room temperature, cells were rinsed twice with 4C ice-cold PBS and lysed for 10 min at 4C. After sonication, 20 l of the lysate were used as DNA input control. The remaining lysate was diluted 10-fold with chromatin immunoprecipitation (ChIP) dilution buffer followed by incubation with the anti-NF-B p65 antibody (Santa-Cruz Biotechnology) or the anti-phospho-STAT1 (Tyr701) antibody (Cell Signaling Technology) overnight at 4C. Immunoprecipitated complexes were collected using protein A/G Plus-agarose beads (Santa-Cruz Biotechnology). The precipitates were extensively washed and then incubated in the elution buffer (1% SDS and 0.1 M NaHCO3) at room temperature for 15 min. Cross-linking of protein-DNA complexes was reversed at 65C for 4 h. DNA was extracted with the Qiagen PCR purification kit. ChIP assays addressing NF-B used the PCR primers 5-CAAGCCAGGGTATGTGGTTT-3 (forward) and 5-GCAGCAGCCATCAGGTATTT-3 (reverse), resulting in a 290-bp fragment. ChIP assays for.The fact that inhibiting Hsp90 prevented iNOS expression in cells and postischemic myocardium suggest that Hsp90 may be a useful target to intervene NO production in diseases such as myocardium infarction. GRANTS This ongoing work was supported by National Heart, Lung, Blood Institute Grants HL-77575 and HL-86965. appearance was attenuated by Hsp90 inhibition in vivo markedly. Intriguingly, additional analyses demonstrated that inhibiting Hsp90 acquired no significant influence on the activation of either IKK-NF-B or JAK-STAT1 in LPS/IFN–stimulated cells. Neither was the nuclear transportation of energetic NF-B or STAT1 suffering from Hsp90 inhibition. But Hsp90 inhibition markedly decreased the binding of energetic NF-B and STAT1 with their DNA components. Chromatin immunoprecipitation assays verified that Hsp90 was needed for NF-B and STAT1 bindings to iNOS promoters inside cells. These research show that besides performing as an allosteric enhancer, Hsp90 can be necessary for transcriptional aspect binding amid iNOS mRNA transcription. Because of the fundamental function of Hsp90 in iNOS gene transactivation, concentrating on Hsp90 may signify a new method of intervene iNOS appearance in illnesses. for 15 min, as well as the supernatant was retrieved. Protein concentrations had been dependant on using the detergent-compatible proteins assay package (Bio-Rad). The proteins had been separated by SDS-PAGE, used in nitrocellulose membranes, and probed with the correct principal antibodies. Membrane-bound principal antibodies had been detected with supplementary antibodies conjugated with horseradish peroxidase. Immunoblots had been developed on movies using the improved chemiluminescence technique (SuperSignal Western world Pico, Pierce). RT-PCR. Total RNA of cultured cells of cardiac tissue had been extracted through the use of TRIzol Reagent (Invitrogen) based on the manufacturer’s guidelines. Change transcription was completed with the Great Capacity cDNA Change Transcription Package (Applied Biosystems). PCR was performed with Taq DNA polymerase. The next primers had been used for discovering iNOS: 5-GGGATGGCTTGCCCCTGG-3 and 5-CGGAGGCAGCACATCAAAG-3. Primers 5-GGTGAAGGTCGGAGTCAACG-3 and 5-CAAAGTTGTCATGGATGACC-3 had been used for calculating GAPDH. NF-B and STAT1 binding assays. The nuclei had been extracted from cells by initial incubating them in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4C for 15 min. Following the cells had been homogenized within a course douncer (15 strokes), cell homogenates had been spun at 3,000 for 5 min. The pellets had been retrieved, extensively cleaned, and resuspended in the nuclear removal buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, and 5 mM sodium pyrophosphate, protease inhibitors). The NF-B and STAT1 binding activity of nuclear ingredients had been measured using the TransFactor NF-B colorimetric package (Clontech, Mountain Watch) as well as the DuoSet mouse energetic STAT1 binding package (R&D Systems, Minneapolis), respectively, based on the manufacturer’s education. Chromatin immunoprecipitation. Organic 264.7 cells were treated with Methylnitronitrosoguanidine LPS (2 g/ml) or IFN- (100 U/ml) for 1 h in the existence and lack of geldanamycin. Formaldehyde (1%) was put into the culture moderate, and after incubation over the rocker for 10 min at area temperature, cells had been rinsed double with 4C ice-cold PBS and lysed for 10 min at 4C. After sonication, 20 l from the lysate had been utilized as DNA insight control. The rest of the lysate was diluted 10-fold with chromatin immunoprecipitation (ChIP) dilution buffer accompanied by incubation using the anti-NF-B p65 antibody (Santa-Cruz Biotechnology) or the anti-phospho-STAT1 (Tyr701) antibody (Cell Signaling Technology) right away at 4C. Immunoprecipitated complexes had been collected using proteins A/G Plus-agarose beads (Santa-Cruz Biotechnology). The precipitates had been extensively washed and incubated in the elution buffer (1% SDS and 0.1 M NaHCO3) at area temperature for 15 min. Cross-linking of protein-DNA complexes was reversed at 65C for 4 h. DNA was extracted using the Qiagen PCR purification package. ChIP assays handling NF-B utilized the PCR primers 5-CAAGCCAGGGTATGTGGTTT-3 (forwards) and 5-GCAGCAGCCATCAGGTATTT-3 (invert), producing a 290-bp fragment. ChIP assays for turned on STAT1 binding to its IFN–regulated transcription aspect STAT1 (GAS) site over the iNOS promoter utilized Methylnitronitrosoguanidine primers 5-ACACGAGGCTGAGCTGACTT-3 (forwards) and 5-CACACATGGCATGGAATTTT-3 (change), producing a 186-bp fragment (24). The causing item was separated by 2% agarose gel electrophoresis. Nitrite assay. Total nitrite released in cell lifestyle medium was assessed using a Griess reagent package (Invitrogen). The response contains 20 l of Griess Reagent, 150 l of moderate, and 130 l of deionized drinking water. After incubation from the mix for 30 min at area temperature, nitrite amounts had been assessed at 548 nm utilizing a M2 spectrophotometric microplate audience (Molecular Gadgets). In vivo myocardial infarction in mice. C57BCL/6 mice had been bought from Charles River Laboratories. Mice had been maintained within a pathogen-free environment, and tests on mice had been conducted based on the protocols accepted by the School pet ethics committee. Mice had been anesthetized with ketamine (55 mg/kg) plus xylazine (15.Proc Natl Acad Sci USA 96: 11507C11512, 1999 [PMC free of charge content] [PubMed] [Google Scholar] 8. the binding of energetic NF-B and STAT1 with their DNA components. Chromatin immunoprecipitation assays verified that Hsp90 was essential for NF-B and STAT1 bindings to iNOS promoters inside cells. These studies uncover that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may symbolize a new Methylnitronitrosoguanidine approach to intervene iNOS expression in diseases. for 15 min, and the supernatant was recovered. Protein concentrations were determined by using the detergent-compatible protein assay kit (Bio-Rad). The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the appropriate main antibodies. Membrane-bound main antibodies were detected with secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique (SuperSignal West Pico, Pierce). RT-PCR. Total RNA of cultured cells of cardiac tissues were extracted by using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed with Taq DNA polymerase. The following primers were utilized for detecting iNOS: 5-GGGATGGCTTGCCCCTGG-3 and 5-CGGAGGCAGCACATCAAAG-3. Primers 5-GGTGAAGGTCGGAGTCAACG-3 and 5-CAAAGTTGTCATGGATGACC-3 were utilized for measuring GAPDH. NF-B and STAT1 binding assays. The nuclei were extracted from cells by first incubating them in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4C for 15 min. After the cells were homogenized in a class douncer (15 strokes), cell homogenates were spun at 3,000 for 5 min. The pellets were recovered, extensively washed, and resuspended in the nuclear extraction buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, and 5 mM sodium pyrophosphate, protease inhibitors). The NF-B and STAT1 binding activity of nuclear extracts were measured with the TransFactor NF-B colorimetric kit (Clontech, Mountain View) and the DuoSet mouse active STAT1 binding kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s training. Chromatin immunoprecipitation. RAW 264.7 cells were treated with LPS (2 g/ml) or IFN- (100 U/ml) for 1 h in the presence and absence of geldanamycin. Formaldehyde (1%) was added to the culture medium, and after incubation around the rocker for 10 min at room temperature, cells were rinsed twice with 4C ice-cold PBS and lysed for 10 min at 4C. After sonication, 20 l of the lysate were used as DNA input control. The remaining lysate was diluted 10-fold with chromatin immunoprecipitation (ChIP) dilution buffer followed by incubation with the anti-NF-B p65 antibody (Santa-Cruz Biotechnology) or the anti-phospho-STAT1 (Tyr701) antibody (Cell Signaling Technology) immediately at 4C. Immunoprecipitated complexes were collected using protein A/G Plus-agarose beads (Santa-Cruz Biotechnology). The precipitates were extensively washed and then incubated in the elution buffer (1% SDS and 0.1 M NaHCO3) at room temperature for 15 min. Cross-linking of protein-DNA complexes was reversed at 65C for 4 h. DNA was extracted with the Qiagen PCR purification kit. ChIP assays addressing NF-B used the PCR primers 5-CAAGCCAGGGTATGTGGTTT-3 (forward) and 5-GCAGCAGCCATCAGGTATTT-3 (reverse), resulting in a 290-bp fragment. ChIP assays for activated STAT1 binding to its IFN–regulated transcription factor STAT1 (GAS) site around the iNOS promoter used primers 5-ACACGAGGCTGAGCTGACTT-3 (forward) and 5-CACACATGGCATGGAATTTT-3 (reverse), resulting in a 186-bp fragment (24). The producing product was separated by 2% agarose gel electrophoresis. Nitrite assay. Total nitrite released in cell culture Methylnitronitrosoguanidine medium was measured with a Griess reagent kit (Invitrogen). The reaction consisted of 20 l of Griess Reagent, 150 l of medium, and 130 l of deionized water. After incubation of the combination for 30 min at room temperature, nitrite levels were measured at 548 nm using a M2 spectrophotometric microplate reader (Molecular Devices). In vivo myocardial infarction in mice. C57BCL/6 mice were purchased from Charles River Laboratories. Mice were maintained in a pathogen-free environment, and experiments on mice were conducted according to the protocols approved by the University or college animal ethics committee. Mice were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg). Animals were orally intubated with PE-90 tubing and connected to a mouse mini-ventilator (model 845; Harvard Apparatus). Core body temperature was maintained at 37C with a thermo heating pad and monitored with the rectal thermometer. A.The infarcted myocardium is excised and frozen in liquid nitrogen until iNOS protein and mRNA analyses. Statistics. Neither was the nuclear transport of active NF-B or STAT1 affected by Hsp90 inhibition. But Hsp90 inhibition markedly reduced the binding of active NF-B and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was essential for NF-B and STAT1 bindings to iNOS promoters inside cells. These studies uncover that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may symbolize a new approach to intervene iNOS expression in diseases. for 15 min, and the supernatant was recovered. Protein concentrations were determined by using the detergent-compatible protein assay kit (Bio-Rad). The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the appropriate primary antibodies. Membrane-bound primary antibodies were detected with secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique (SuperSignal West Pico, Pierce). RT-PCR. Total RNA of cultured cells of cardiac tissues were extracted by using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed with Taq DNA polymerase. The following primers were used for detecting iNOS: 5-GGGATGGCTTGCCCCTGG-3 and 5-CGGAGGCAGCACATCAAAG-3. Primers 5-GGTGAAGGTCGGAGTCAACG-3 and 5-CAAAGTTGTCATGGATGACC-3 were used for measuring GAPDH. NF-B and STAT1 binding assays. The nuclei were extracted from cells by first incubating them in hypotonic buffer (10 mM TrisHCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4C for 15 min. After the cells were homogenized in a class douncer (15 strokes), cell homogenates were spun at 3,000 for 5 min. The pellets were recovered, extensively washed, and resuspended in the nuclear extraction buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, and 5 mM sodium pyrophosphate, protease inhibitors). The NF-B and STAT1 binding activity of nuclear extracts were measured with the TransFactor NF-B colorimetric kit (Clontech, Mountain View) and the DuoSet mouse active STAT1 binding kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s instruction. Chromatin immunoprecipitation. RAW 264.7 cells were treated with LPS (2 g/ml) or IFN- (100 U/ml) for 1 h in the presence and absence of geldanamycin. Formaldehyde (1%) was added to the culture medium, and after incubation on the rocker for 10 min at room temperature, cells were rinsed twice with 4C ice-cold PBS and lysed for 10 min at 4C. After sonication, 20 l of the lysate were used as DNA input control. The remaining lysate was diluted 10-fold with chromatin immunoprecipitation (ChIP) dilution buffer followed by incubation with the anti-NF-B p65 antibody (Santa-Cruz Methylnitronitrosoguanidine Biotechnology) or the anti-phospho-STAT1 (Tyr701) antibody (Cell Signaling Technology) overnight at 4C. Immunoprecipitated complexes were collected using protein A/G Plus-agarose beads (Santa-Cruz Biotechnology). The precipitates were extensively washed and then incubated in the elution buffer (1% SDS and 0.1 M NaHCO3) at room temperature for 15 min. Cross-linking of protein-DNA complexes was reversed at 65C for 4 h. DNA was extracted with the Qiagen PCR purification kit. ChIP assays addressing NF-B used the PCR primers 5-CAAGCCAGGGTATGTGGTTT-3 (forward) and 5-GCAGCAGCCATCAGGTATTT-3 (reverse), resulting in a 290-bp fragment. ChIP assays for activated STAT1 binding to its IFN–regulated transcription factor STAT1 (GAS) site on the iNOS promoter used primers 5-ACACGAGGCTGAGCTGACTT-3 (forward) and 5-CACACATGGCATGGAATTTT-3 (reverse), resulting in a 186-bp fragment (24). The resulting product was separated by 2% agarose gel electrophoresis. Nitrite assay. Total nitrite released in cell culture medium was measured with a Griess reagent kit (Invitrogen). The reaction consisted of 20 l of Griess Reagent, 150 l of medium, and 130 l of deionized water. After incubation of the mixture for 30 min at room temperature, nitrite levels were measured at 548 nm using a M2 spectrophotometric microplate reader (Molecular Devices). In vivo myocardial infarction in mice. C57BCL/6 mice were purchased from Charles River Laboratories. Mice were maintained in a pathogen-free environment, and experiments on mice were conducted according to the protocols approved Col13a1 by the University animal ethics committee. Mice were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg). Animals were orally intubated with PE-90 tubing and connected to a mouse mini-ventilator (model 845; Harvard Apparatus). Core body temperature was taken care of at 37C having a thermo heating pad and.