All animal work was approved by the University of Rochester University Committee on Animal Resources (UCAR) committee (UCAR 2007-065). If a mouse showed that it had aspirated fluid or significant body weight loss (10% or more), and did not die immediately, the mouse was humanely euthanized.
Bacterial strains and growth condition
Bacterial strains used in this study included Salmonella typhimurium
wild-type strain ATCC14028 (WT-SL) and non-pathogenic Salmonella
mutant strain PhoPc
. Non-agitated microaerophilic bacterial cultures were prepared as previously described 
Human colonic epithelial HCT116, CaCo2BBE, and HT29C19A cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin and L-glutamine. The rat small intestinal IEC-18 cell line was grown in DMEM (high glucose, 4.5 g/L) containing 5% FBS (vol/vol), 0.1 U/ml insulin, 50 µg/ml streptomycin, and 50 U/ml penicillin.
Streptomycin pre-treated mouse model
Animal experiments were performed using specific-pathogen-free female C57BL/6 mice (Taconic, Hudson, NY) that were 6–7 weeks old, as previously described 
. The protocol was approved by the University of Rochester University Committee on Animal Resources (UCAR). Water and food were withdrawn 4 hours before oral gavage with 7.5 mg/mouse of streptomycin (100 µl of sterile solution or 100 µl of sterile water in control). Afterwards, animals were supplied with water and food ad libitum. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 hours before the mice were infected with 1×107
CFU of S. Typhimurium
(100 µl suspension in HBSS) or treated with sterile HBSS (control). Eight hours after infection, mice were sacrificed, and tissue samples from the intestinal tracts were removed for analysis.
Mouse colonic epithelial cells
Mouse colonic epithelial cells were collected by scraping the mouse colon, including the proximal and distal regions. Cells were sonicated in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, protease inhibitor cocktail). The protein concentration was measured using BioRad Reagent (BioRad, Hercules, CA, USA).
Plasmids with c-myc-tagged, wild-type Axin1, Axin2, and Axin1 mutants were from Dr. Hsu's laboratory. Transient transfections were performed with Lipofectamine2000 (Invitrogen, San Diego, CA) in accordance with the manufacturer's instructions. At the indicated times after transfection, proteins were extracted with RIPA buffer (50 µM Tris-Hcl, pH 8.0 with 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for immunoblotting.
HCT116 cells were grown in 12-well plates. The cells were transfected with on-Target plus smart pool human Axin 1 siRNA (Dharmacon Inc., Lafayette, MO) or scrambled siRNA control (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) using Surefect reagent (SABiosciences, Frederick, MD). After transfection for 72 hours, cells were colonized by Salmonella for 30 min, washed, and incubated for 30 min in DMEM with Gentamicin (500 µg/ml), and then the levels of Axin and β-actin were assessed by western blot.
Mouse epithelial cells were scraped and lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, protease inhibitor cocktail), and then the protein concentration was measured. Cultured cells were rinsed twice in ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-Axin1 (Cell Signal, Beverly, MA, U.S.A), anti-APC, anti-Villin,anti-c-Myc (Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.), anti-ubiquitin (Enzo life Science, 5120 Butler Pike Plymouth Meeting, PA, U.S.A.), anti-sumo1 (Boston Biochem, Cambridge, MA, USA), anti-GSK-3β, anti-β-catenin (1
1000; BD, San Jose, CA, U.S.A.), or anti-β-actin (Sigma-Aldrich, Milwaukee, WI, U.S.A.) antibodies and were visualized by ECL. Membranes that were probed with more than one antibody were stripped before re-probing.
Cells were rinsed twice in ice-cold HBSS and lysed in cold immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris·HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate) containing protease inhibitor cocktail. Samples were precleared with protein A-agarose. Pre-cleared lysates were incubated with 2 µg of primary antibodies for 1 hour at 4°C. Protein A-agarose was added to the lysate and incubated for 30 min with agitation at 4°C before being washed with cold immunoprecipitation buffer. The pellet was resuspended in 0.1 M glycine, pH 2.5, and incubated with agitation for 10 min at 4°C and then centrifuged at 9,000 g for 2 minutes. The supernatant was removed and neutralized with 1 M Tris·HCl, pH 8.0. The samples were diluted with concentrated (5×) electrophoresis sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol), boiled for 5 minutes, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. Membrane blots were probed with secondary antibody and visualized by ECL.
Colonic tissues from the proximal and distal portion of the colon were freshly isolated and paraffin-embedded after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (1 µm) of mouse colons. After preparation of the slides as described previously 
, slides were incubated in 3% hydrogen peroxide for 20 minutes at room temperature to block endogenous peroxidase activity, followed by incubation for 20 minutes in 5% BSA with 0.1% saponin in PBS to reduce nonspecific background.
Cultured cells were rinsed three times in HBSS, fixed for 20 minutes in 100% cold ETOH, and permeabilized for 10 minutes with 0.2% Triton X-100, followed by three rinses with Hanks HBSS, and incubation for 30 minutes in 10% BSA in HBSS to reduce nonspecific background. The permeabilized cells or tissue samples were incubated with primary antibodies for 90 minutes at room temperature. Samples were then incubated with goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 594 (Molecular Probes, CA; 1
200) and DAPI (Molecular Probes 1
10,000) for 1 hour at room temperature. Tissues or cells were mounted with SlowFade (SlowFade® AntiFade Kit, Molecular Probes), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Leica SP5 Laser Scanning confocal microscope.
S. typhimurium invasion of human epithelial monolayers
Infection of HCT116 cells was performed by a previously described method 
. Bacterial solution was added, and bacterial invasion was assessed after 30 minutes. Cell-associated bacteria, representing bacteria adhered to and/or internalized into the monolayers, were released by incubation with 100 µl 1% Triton X-100 (Sigma). Internalized bacteria were those obtained from lysis of the epithelial cells with 1% Triton X-100 30 minutes after the addition of gentamicin (50 µg/ml). For both cell-associated and internalized bacteria, 0.9 ml LB broth was added, and each sample was vigorously mixed and quantitated by plating for CFU on MacConkey agar medium.
Salmonella-induced human IL-8 secretion
HCT116 cells were cultured in DMEM, followed by Salmonella-containing HBSS for 30 minutes, washed 3 times in HBSS, and incubated at 37°C for 6 hours. Cell supernatants were removed and assayed for IL-8 by ELISA in 96-well plates.
Real Time quantitative PCR
Total RNA was extracted from epithelial cell monolayers or mouse colonic epithelial cells using TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA integrity was verified by gel electrophoresis. RNA reverse transcription was done using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's directions. The RT-cDNA reaction products were subjected to quantitative real-time PCR using the MyiQ single-color real-time PCR detection system (Bio-Rad) and iQ SYBR green supermix (Bio-Rad) according to the manufacturer's directions. All expression levels were normalized to β-actin levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCR reactions were performed in triplicate. All PCR primers were designed using Lasergene software () (DNAStar, Madison, WI).
Data are expressed as mean ± SD. Differences between two samples were analyzed by Student's t test. P-values of 0.05 or less were considered statistically significant. Differences among three or more groups were analyzed using ANOVA (SAS 9.2 version, SAS Institute Inc., Cary, NC).