Western blot detection of phosphorylation of EGFR, Akt, PKCα, and Hsp72.

<p>(A) Representative panels of p-EGFR, total-EGFR, p-Akt, total-Akt, and p-PKCα proteins in IPEC-J2 cells collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. (B) The intensities of p-EGFR, total-EGFR, p-Akt, total-Akt, and p-PKCα bands were determined using Quantity One software. Results are presented as the ratio of the p-EGFR band intensity to the total-EGFR band intensity, the ratio of the p-Akt band intensity to the total-Akt band intensity, and the ratio of p-PKCα band intensity to the GAPDH band intensity. (C) Representative panels of heat shock protein 72 (Hsp72), p-EGFR, and p-PKCα in IPEC-J2 cells treated with <i>L</i>. <i>rhamnosus</i> alone for 0, 3, and 10 h. Expression of GAPDH was measured as an internal control. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Incubation with <i>L</i>. <i>rhamnosus</i> reduces adhesion of F4⁺ ETEC to the IPEC-J2 cell monolayer.

<p>IPEC-J2 cells cultured in medium with and without porcine mucin were subjected F4^<b>+</b> ETEC challenge alone (ETEC), co-incubated with <i>L</i>. <i>rhamnosus</i> plus F4^<b>+</b> ETEC challenge simultaneously (<i>LR</i> + ETEC), or pre-incubated with <i>L</i>. <i>rhamnosus</i> for 2 h followed by F4^<b>+</b> ETEC challenge [(–2 h) <i>LR</i> + ETEC]. At 3 h following F4^<b>+</b> ETEC (10^<b>7</b> CFU/ml) challenge, the number of F4^<b>+</b> ETEC CFUs recovered from IPEC-J2 cells was determined (A) and the number of F4^<b>+</b> ETEC CFUs recovered from mucin-coated microtiter plate wells with and without <i>L</i>. <i>rhamnosus</i> incubation was determined (B). Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Concentrations of TNF-α, IL-10, and PGE₂ in IPEC-J2 cell culture supernatants.

<p>Supernatants were collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. The concentrations of (A) TNF-α, (B) IL-10, and (C) PGE_<b>2</b> were determined by ELISA. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of <i>L</i>. <i>rhamnosus</i> on apoptosis of IPEC-J2 cells.

<p>IPEC-J2 cells were collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. Apoptosis was assessed by flow cytometry. (A) Representative two-dimensional scatter plots of annexin V versus propidium iodide. (B) The percentage of early apoptotic cells. (C) The percentage of late apoptotic cells. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of <i>L</i>. <i>rhamnosus</i> on the mucin layer.

<p>(A) IPEC-J2 cells were cultured with medium alone (CONT), F4^<b>+</b> ETEC (ETEC), <i>L</i>. <i>rhamnosus</i> (<i>LR</i>), <i>L</i>. <i>rhamnosus</i> plus F4^<b>+</b> ETEC simultaneously (<i>LR</i> + ETEC), or pre-incubated with <i>L</i>. <i>rhamnosus</i> for 2 h followed by F4^<b>+</b> ETEC challenge [(–2 h) <i>LR</i> + ETEC]. Mucin production (purple clusters, arrows) was determined by AB-PAS staining of the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. (B) Semi-quantitative determination of mucin (purple) production. Digital images were analyzed using Image Pro Plus 6.0 software, which enabled quantification of mucin level as the mean of integrated optical density (IOD). The ratio of purple mucin IOD to blue mucin IOD was calculated. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. Scale bars, 100 μm.</p

Treatment with <i>L</i>. <i>rhamnosus</i> alters <i>TLR</i> and <i>NLR</i> expression after F4⁺ ETEC infection.

<p>IPEC-J2 cells were collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. The relative expression of mRNAs for genes encoding (A) <i>TLR2</i>, (B) <i>TLR4</i>, (C) <i>TLR9</i>, (D) <i>NOD1</i>, and (E) <i>NOD2</i> was analyzed by quantitative real-time PCR. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Western blot detection of tight junction proteins.

<p>(A) Representative panels of zonula occludens-1 (ZO-1) and occludin proteins in IPEC-J2 cells collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. Expression of GAPDH was measured as an internal control. Results are presented as the ratio of the (B) ZO-1 band intensity and (C) occludin band intensity to the GAPDH band intensity. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

MOESM1 of Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Additional file 1. Information of oligonucleotide primers used for quantitative real-time PCR. The table shows the sequences of primers used for real-time PCR, length of the respective PCR product and gene accession number in this study

MOESM2 of Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Additional file 2. Effect of orally fed BLS-mix on faecal Escherichia shedding before and after E. coli infection. Fresh faecal samples from animals of the indicated groups were collected on days 1, 4, 7, 9 and 12 after weaning. Bacterial DNA isolated from 200 mg of faeces from pigs among four groups was analyzed by quantitative PCR using universal primers for Escherichia 16S rRNA gene. Results are presented as log10 copies/g faeces. Data are presented as means ± SEM (n = 5 per group). Mean values at the same time point without a common superscript letter differ significantly

Additional file 6: of Oral administration of a select mixture of Bacillus probiotics generates Tr1 cells in weaned F4ab/acR− pigs challenged with an F4+ ETEC/VTEC/EPEC strain

Immunofluorescence staining of F4 + ETEC/VTEC/EPEC strain in the ileum. The figures show representative photomicrographs of F4+ ETEC/VTEC/EPEC (red) adhesion to the ileal mucosa of pigs 1 week after F4+ ETEC/VTEC/EPEC challenge. The typical features associated with an attachment rating were as follows: 0 (A), no observed attachment of F4+ ETEC/VTEC/EPEC to the ileal mucosa; 1 (B); 2 (C); 3 (D); and 4 (E), F4+ ETEC/VTEC/EPEC were adhered to the crypt and entire villus. Scale bars, 50 μm