*4.6. Quantifying Adhe7rent Bacteria*

To calculate the number of adherent *E. coli*, *E. coli* were cocultured with IPEC-J2 cells in DMEM/high-glucose medium (no penicillin/streptomycin, no FBS). Then, the cells were washed with PBS six times and lysed with 1% Triton X-100 for 20 min at room temperature. Next, 5 μL lysates were plated on MacConkey agar plates overnight. The total number of bacteria was quantified as CFUs.

### *4.7. TER and Permeability Measurement*

To evaluate the barrier integrity of IPEC-J2 cells, the transepithelial resistance (TER) and was measured, and a permeability assay was performed. The initial TER was tested before the cells were seeded. IPEC-J2 cells were then seeded in a Transwell membrane insert (12 mm diameter, 0.4 μm pore size, Corning) at a density of 7 × 105 cells/well. Then, 200 <sup>μ</sup>L and 500 <sup>μ</sup>L medium was added to the apical and basal compartments, respectively. Cecropin A (12.5 μg/mL) was added to the apical and basal compartments. The TER values were measured every day by using an ohm-meter fitted with chopstick electrodes (Millipore ESR-2; Burlington, MA, USA). Before each test, the plates were placed at room temperature for 30 min. The TER was calculated by using the following equation:

$$\text{TER (\$\Omega\$-cm}^2\$) = (\text{TER } - \text{TER}\_{\text{initial}}) \times 0.34$$

To evaluate the permeability of the monolayer intestinal cells, FITC-dextran was used. FITC-dextran (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in PBS at 5 mg/mL. After the medium was discarded, 200 μL FITC-dextran was added to the apical compartment, and 500 μL PBS was added to the basal compartment and cultured for 2 h. Then, 100 μL liquid from each well was transferred to 96-well plates, and the absorbance was read at 480 nm excitation and 520 nm emission wavelengths. Then, the content of the FITC-dextran in the basal compartment was calculated using a standard curve.

#### *4.8. qPCR*

Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Concentration and purity of RNA was checked by using a NanoDrop 2000. cDNA was generated from 1 μg total RNA using a First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Quantitative PCR (qPCR) was performed to quantify mRNA expression levels of IL-6, IL-8 and TNF-α relative to that of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using SYBR Green mix (ABI) according to the manufacturer's instructions. The forward and reverse primers are shown in Table S3.

### *4.9. Western Blotting*

The total protein was extracted with lysis buffer. The concentration of protein was tested by using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) and mixed with 5× loading buffer. 20 μg protein sample was loaded in each well. The supernatant was then separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Burlington, MA, USA). After blocking with 5% skimmed milk powder, the membrane was incubated with the appropriate primary antibodies overnight at 4 ◦C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h. Bands were detected using an ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). Band intensity was quantified using ImageJ software. The primary antibodies including β-actin, p-MEk, MEK, p-ERK, ERK, CDX2 were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). To specifically inhibit the phosphorylation of ERK, PD184352 (CST) was dissolved in DMSO and then used at a concentration of 10 μM for 48 h.

#### *4.10. Cell Immunofluorescence Aassay*

To test the expression and location of tight junction proteins (ZO-1, occludin and claudin-1) [31] and the cytoskeleton (F-actin), cell immunofluorescence was used. The IPEC-J2 cells were cultured and became confluent on the slide. After treatment, the cells were washed with PBS three times, fixed with 4% polyoxymethylene for 30 min, and then washed by PBS again three times. For F-actin staining, 0.5% Triton X-100 was added for 20 min. After that, 0.5% bovine serum albumin was added for 1 h, and then the primary antibodies were added and incubated with the cells at 4 ◦C overnight. The slides were then washed with PBST three times, and the FITC-labeled secondary antibody was added for 2 h. At last, the cell nuclei were stained by using DAPI (Santa Cruz Biotechnology, Dallas, TX, USA). The slides were then observed by using a fluorescence microscope. Primary antibodies including occludin (Abcam, Cambridge, UK), claudin-1 (CST, Danvers, MA, USA) and ZO-1 (Thermo Fisher Scientific, Waltham, MA, USA) were used. For staining F-actin, FITC-phalloidin (Sigma-Aldrich, St. Louis, MI, USA) was used.

### *4.11. Statistics*

Data are expressed as the mean ± SEM. The Student's *t*-test was conducted to determine the differences between 2 groups using SAS (version 9.2, SAS Institute Inc., Cary, NC, USA), and a one-way ANOVA was used to determine differences among groups. Differences were considered statistically significant when *p* < 0.05.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/1941/ s1.

**Author Contributions:** Study concept and design: Z.Z.; data acquisition: Z.Z., X.N., J.L. and C.J.; W.R. provided the bacteria strain for this study; Z.Z., J.D., B.D. and Y.Y. wrote and modify the manuscript.

**Funding:** The authors gratefully thank the Nation K&D Program of China (2018YFD0500603), the National Natural Science Foundation of China (Grant Nos. 31790411) and the Natural Science Foundation of Guangdong Province (Grant Nos. 2017A030310410 and 2017A030310398) for supporting of the projects. The studies meet with the approval of the university's review board.

**Conflicts of Interest:** The authors declare no conflicts of interests.
