Bindloss et al Nikon DAPl-FITC-tRITC triple excitation filter set. An LED lamp (Lumencor, Beaverton OR) was used for illumination. 16-bit images were captured by the Zyla SCMOS camera (Concord, MA)and processed by the Nis-Elements imaging software Western blot Total protein was extracted from Caco-2 cells with lysis buffer(50mM Tris-HCl pH 7.6, 150mM NaCl, 0. 1%v/v NP-40, ImM EDTA)mixed with protease inhibitor( Calbiochem) Bradford assay was conducted for protein concentration, and 9 ug were loaded and separated by a 6% SDS-PAGE and transferred onto a Immobilon-P PVDF transfer membrane (Millipore Sigma) Membranes were further probed with antibodies against iNOS (2ug/ml R&D)or alpha/beta tubulin(1: 200, Cell Signaling. Proteins of interest were detected with HRP-conjugated goat anti-mouse IgG antibody(1: 1000, Jackson) and donkey anti-rabt IgG(1: 1000, Jackson)and visualized with Supersignal West Pico Chemiluminescent Substrate(Termoscientific)according to manufacturers'instructions. All experiments were repeated at least four times independently Nitrate measurements Intestinal nitrate measurements were performed as described previously (30). Briefly, uninfected mice were euthanized and the intestine was removed and divided along its sagittal plane. The mucus layer was gently scraped from the tissue and homogenized in 200 ul PBS and then placed on ice. Samples were centrifuged at 5,000 x g for 10 min at 4C to emove the remaining solid particles. The supernatant was then filter sterilized (0.2-um Acrodisc syringe filter, Pall Life Sciences). Measurement of intestinal nitrate followed adaptation of the Griess assay In this assay, nitrate was first reduced to nitrite by combining 50 ul of each sample with 50 ul of Griess reagent I containing vanadium(lIn) chloride(0.5 M HCI, 0.2 mM VCl3, 1%sulfanilamide), and then the mixture was incubated at room temperature for 10 min. Next, 50 ul of Griess reagent 2 [0. 1%(1-naphthyl )ethylenediamine dichloride] was added to each sample. Absorbance at 540 nm was measured immediately fter the addition of Griess reagent 2 to detect any nitrite present in the samples. The samples were then incubated for 8 h at room temperature( to allow for reduction of nitrate to nitrite), and the absorbance at 540 nm was measured again. The initial absorbance(prior to reducing nitrate to nitrite) was subtracted from the absorbance after 8 h to determine nitrate concentrations in the cecal mucus layer Samples were tested in duplicate, and al measurements were standardized to the initial sample weight. To measure nitrate for in vitro assays, polarized Caco-2 cell medium was changed to MEM containing 10% fetal bovine serum(FBs)medium without phenol, the day before experiment. At 24 hours post all supernatant was collected, and nitrate was measured using the griess assa as described above Nitrate reductase activity assay Triplicate aerobic overnight cultures grown in Luria-Bertani (lB) Broth with or without appropriate antibiotic depending on strain were diluted in fresh lB Broth with 40 mM NaNO3 and incubated for 3 hours at 37C without shaking in closed tubes. After incubation, cultures were placed on ice, an aliquot was transferred to a microcentrifuge tube and lleted. Supernatant was removed and the cells were resuspended in Phosphate Buffered Science Author manuscript; available in PMC 2017 October 1Nikon DAPI-FITC-TRITC triple excitation filter set. An LED lamp (Lumencor, Beaverton, OR) was used for illumination. 16-bit images were captured by the Zyla sCMOS camera (Concord, MA) and processed by the NIS-Elements imaging software. Western Blot Total protein was extracted from Caco-2 cells with lysis buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 0.1% v/v NP-40, 1mM EDTA) mixed with protease inhibitor (Calbiochem). Bradford assay was conducted for protein concentration, and 9 μg were loaded and separated by a 6% SDS-PAGE and transferred onto a Immobilon-P PVDF transfer membrane (Millipore Sigma). Membranes were further probed with antibodies against iNOS (2ug/ml, R&D) or alpha/beta tubulin (1:200, Cell Signaling). Proteins of interest were detected with HRP-conjugated goat anti-mouse IgG antibody (1:1000, Jackson) and donkey anti-rabbit IgG (1:1000, Jackson) and visualized with Supersignal West Pico Chemiluminescent Substrate (Termoscientific) according to manufacturers’ instructions. All experiments were repeated at least four times independently. Nitrate measurements Intestinal nitrate measurements were performed as described previously (30). Briefly, uninfected mice were euthanized, and the intestine was removed and divided along its sagittal plane. The mucus layer was gently scraped from the tissue and homogenized in 200 μl PBS and then placed on ice. Samples were centrifuged at 5,000 × g for 10 min at 4°C to remove the remaining solid particles. The supernatant was then filter sterilized (0.2-μm Acrodisc syringe filter, Pall Life Sciences). Measurement of intestinal nitrate followed an adaptation of the Griess assay. In this assay, nitrate was first reduced to nitrite by combining 50 μl of each sample with 50 μl of Griess reagent 1 containing vanadium(III) chloride (0.5 M HCl, 0.2 mM VCl3, 1% sulfanilamide), and then the mixture was incubated at room temperature for 10 min. Next, 50 μl of Griess reagent 2 [0.1% (1-naphthyl)ethylenediamine dichloride] was added to each sample. Absorbance at 540 nm was measured immediately after the addition of Griess reagent 2 to detect any nitrite present in the samples. The samples were then incubated for 8 h at room temperature (to allow for reduction of nitrate to nitrite), and the absorbance at 540 nm was measured again. The initial absorbance (prior to reducing nitrate to nitrite) was subtracted from the absorbance after 8 h to determine nitrate concentrations in the cecal mucus layer. Samples were tested in duplicate, and all measurements were standardized to the initial sample weight. To measure nitrate for in vitro assays, polarized Caco-2 cell medium was changed to MEM containing 10% fetal bovine serum (FBS) medium without phenol, the day before experiment. At 24 hours post treatments, cell supernatant was collected, and nitrate was measured using the Griess assay as described above. Nitrate reductase activity assay Triplicate aerobic overnight cultures grown in Luria-Bertani (LB) Broth with or without appropriate antibiotic depending on strain were diluted in fresh LB Broth with 40 mM NaNO3 and incubated for 3 hours at 37°C without shaking in closed tubes. After incubation, cultures were placed on ice, an aliquot was transferred to a microcentrifuge tube and pelleted. Supernatant was removed and the cells were resuspended in Phosphate Buffered Byndloss et al. Page 12 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript