S1 The primer used in this research

Table the primer used in this research

Gene name / Primer(5’→3’) / GenBank accession no. / Product (bp) / References
β-actin / F: GGACCTGACCGACTACCTCAT
R: GGGCAGCTCGTAGCTCTTCT / DQ452569 / 181 / (Zhu et al., 2013)
B2M / F: TTCACACCGCTCCAGTAG
R: CCAGATACATAGCAGTTCAGG / NM_213978 / 166 / (Zhu et al., 2013)
Fas / F: TGATGCCCAAGTGACTGACC
R: GCAGAATTGACCCTCACGAT / NM_213839 / 103 / (Zhu et al., 2013)
Bcl-2 / F: ACCTGAATGACCACCTAGAGC
R: TCCGACTGAAGAGCGAAC / NM_214285 / 180 / (Zhu et al., 2013)
P53 / F:CTGCTTCCTGAAAACAACC
R:AAGGGACAAAGGACGACA / NM_213824 / 199 / (Zhu et al., 2012)
Caspase-3 / F: GTGGGACTGAAGATGACA
R: ACCCGAGTAAGAATGTG / NM_214131 / 190 / (Zhu et al., 2013)
Caspase-8 / F: AGACAAGGGCATCATCATCGG
R: GGTTTACCAAGAAGGGAACGG / NM_001031779 / 102 / (Zhu et al., 2013)
Caspase-9 / F: AATGCCGATTTGGCTTACGT
R: CATTTGCTTGGCAGTCAGGTT / XM_003127618 / 195 / (Zhu et al., 2013)

Reference

Zhu, L. et al. 2013. Effect of N-acetyl cysteine on enterocyte apoptosis and intracellular signalling pathways' response to oxidative stress in weaned piglets. Br. J. Nutr. 110: 1938-1947.

Zhu, L. H., K. L. Zhao, X. L. Chen, and J. X. Xu. 2012. Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs. J. Anim. Sci. 90: 2581-2589.

S2 The evidence that IPEC-J2 cells retain original functions of primary cells

Many evidences showed that IPEC-J2 cells cultured for a long time still retain original functions of primary cells, such as express keratin-18 (which is a biomarker of epithelial cell), can express secretes mucins, produces cytokines/chemokines, and expresses Toll-like receptors. We tested these item and show it below to confirm the character of IPEC-J2 we used this study:

Part 1 Immunofluorescence for keratin-18 by IPEC-J2 cells

Fig. S2-1 Immunofluorescence for keratin-18 by IPEC-J2 cells (a) keratin-18 primary antibodies + FITC- second antibody; (b) PBS + FITC- second antibody; (c)PBS+PBSInnate immune activation of swine.

Part 2 PCR result show IPEC-J2 cells can express mucins, cytokines/chemokines, and Toll-like receptors

The PCR mixture (20μl) include 2μl cDNA, 10μl Premix TaqTM, 0.4μl sense primer, 0.4μl anti-sense primer and 6.2μl ddH2O. The primer sequences list in table S2-1. The PCR program was performed as pre-denaturation at 95℃for 30 s and forty cycles of 95℃for 5 s, 60℃for 30s and 72℃for 35 s. the PCR products were test by agarose gel electrophoresis and sequenced by Sunny Biotech (Shanghai, China).

Table S2-1 The primer used in this study

Gene name / Primer(5’→3’) / GenBank no. / Product (bp) / References
MUC1 / F:GGGCTTCTGGGACTCTTT / XM_001926883 / 143 / This study
R: AGGTTATAGGTGCCTGCTT
MUC4 / F:TGGGATGATGCTGATTTCT / NM_001206344 / 148 / This study
R:GTGTCCACCTGGCTTTGT
pBD-1 / F: TCCTTGTATTCCTCCTCA / NM-213838 / 93 / [1]
R: ACACGCCTTTATTCCTTA
pBD-2 / F: ACCTGCTTACGGGTCTTG / AY506573 / 168 / [1]
R: CTCTGCTGTGGCTTCTGG
pBD-3 / F: GAAGTCTACAGAAGCCAAAT / AY460575 / 102 / [1]
R: GGTAACAAATAGCACCATAA
lysozyme / F: CTTCTCCTCCTTTCTGTCT / NM_214392.2 / 246 / This study
R: AACTGCTTTGGGTGTCTT
PMAP-37 / F: CTTAGCCGACTGCGTGAT / L39641.1 / 190 / [2]
R: TAGCCTGAATCTTAGGACTGAA
Occludin / F: GCTGGAGGAAGACTGGAT / NM_001163647 / 244 / [3]
R: ATCCGCAGATCCCTTAAC
Claudin-1 / F: AGAAGATGCGGATGGCTGTC / NM_001244539 / 193 / [4]
R: CCCAGAAGGCAGGAGAAGC
ZO-1 / F: ACCCACCAAACCCACCAA / CK_453343 / 123 / [3]
R: CCATCTCTTGCCAAACTATC
TLR-1 / F:TGCTGGATGCTAACGGATGTC
R: AAGTGGTTTCAATGTTGTTCAAAGTC / AB219564 / 102 / [5]
TLR-2 / F:TCACTTGTCTAACTTATCATCCTCTTG
R:TCAGCGAAGGTGTCATTATTGC / AB085935 / 162 / [5]
TLR-3 / F: AGTAAATGAATCACCCTGCCTAGCA
R: GCCGTTGACAAAACACATAAGGACT / DQ266435 / 110 / [5]
TLR-4 / F:GCCATCGCTGCTAACATCATC / AB188301 / 108 / [5]
R:CTCATACTCAAAGATACACCATCGG

The results of agarose gel electrophoresis (Fig S2-2, Fig S2-3) showed the IPEC-J2 cell we cultured can express TLR-1(Toll-like recepter-1), TLR-2, TLR-3, TLR-4, Occludin, Claudin-1, ZO-1, lysozyme, PMAP-37(an antibacterial peptide), pBD-1(an antibacterial peptide that plays an important role in defense system of porcine), pBD-2, pBD-3, MUC1(a kind of mucins) and MUC4. The sequence result confirmed it (Table S2-2).

More evidences such as protein expression please refer to the review by Brosnahan AJ et al6].

Fig. S2-2 Agarose gel electrophoresis of M1: DNA maker; 1: TLR-1; 2: TLR-2; 5: Occludin; 6: Claudin-1; 7: ZO-1; 8: lysozyme.

Fig. S2-2 Agarose gel electrophoresis of M2: DNA maker; 9: PMAP-37; 10: pBD-1; 11: pBD-2; 12: pBD-3; 13: MUC1; 14: MUC4.

Table S2-2 Sequence result

Gene / Sequence / Identify
TLR-1 / ACCCGCAAGTTCATTGAACTTCACTTATCTCATAGTTATCCAACCTGACTTTGACAACATTGAAACCACTTAA / 95%
(NM_001031775)
TLR-2 / GCCAATTGGAAGCTCTCGCTGTACTAACTCTGACCTTACTGGGAAATCCTTACAAAACACTCGGGGAAACACCGCTTTTTTCTCATCTCCCAAATCTGCGAATCCTGAAAATAGGCAATAATGACACCTTCGCTGAA / 100%
Occludin / GTCTCACTTCGATCACAAAGGCACTCTACAAGAGAATTTTGACAGTGGCCTGCAGGAGTACAAGAGCTTACAAGCAGAACTTGATGAGATCAATAAAGAGCTCTCTCGACTGGATAAAGAGCTGGATGATTATAGAGAAGAAAGTGAAGAGTACATGGCTGCTGCTGATGAATACAATAGACTGAAGCAAGTTAAGGGATCTGCGGATA / 98%
Claudin-1 / CATCGCATACTGCGTACTCATGTCTGGCTATCTTAGTTGCCACAGCATGGTATGGTAACAGAATTGTTCAAGAATTCTATGACCCCATGACCCCAGTCAATGCCAGATATGAATTTGGTCAGGCTCTCTTCACTGGCTGGGCTGCTGCTTCTCTCTGCCTTCTGGGAGA / 99%
ZO-1 / AGGGAAGTATCCCGGAAATGAGAATATGGTCTTCGATTGGCCAGCCATATATTTGTAAAGGAAATTTCACAAGATAGTTTGGCAGCAAGAGATGGA / 97%
XM_003480423
lysozyme / AGAAATTCTATTGCTCGGCTGTCGAGTTCGCCAGAATTCTCGAAAAGTCTAGGCATGGACGGCTATAGGGGAGTCAGCCTGGCGAACTGGGTGTGTTTGGCCAAGTGGGAAAGTGATTTTAACACAAAAGCTATAAACCGCAATGTTGGAAGCACTGATTATGGAATATTTCAAATTAATAGCCGCTACTGGTGTAATGATGGCAAGACACCCAAAGCAGTTA / 98%
PMAP-37 / GGTTCTGCTCGGGAGCTTGGTGAAGATTGAAAGAATTGGTCAGAAAATCAAAGATTTATCTGAGTTTTTTCAGTCCTAAGATTCAGGCTA / 94%
pBD-1 / failed
pBD-2 / GAACTTACGTAATATGTGCCAGTAAGGGGGGACCTGCAACTTCTCCCCCTGCCCGCTCTTCAACAGGATTGAAGGGACCTGTTACAGTGGCAAGGCCAAGTGCTGCATCCGCTGACCCTGAGCCAGAAGCCACAGCAGAGA / 98%
pBD-3 / AAAATTTTTTTTGCATTGAAGAGCTTCAGTTATTATTATTATTTTTCAAATTCTGTTTATGGTGCTATTTGTTACCAC / 88%
MUC1 / GGAATGTTAACACTGTACTGGTAGAGCTGATTCTGGCCTTCCAAGACAGTGCCGCTGCCCACAACCTGAAGACACAGTTTGATCGGCTTAAAGTAGAAGCAGGCACCTATAACCTA / 98%
MUC4 / TGACACGTTTACAAGAGTATGAGACGCTCTATGATGAATACAACCAGCTAGTGTGGGAGGTGGAGTATTGGATTAAAATGTTAACAAACACCTGGAACTACAAAGCCAGGTGGACACA / 97%

Refrence

1.Qi, S., et al., β-defensins gene expression in tissues of the crossbred and Tibetan pigs. Livestock Science, 2009. 123(2–3): p. 161-168.

2.Ma, Q.Q., et al., Tissue specificity and species superiority of cathelicidin gene expression in Chinese indigenous Min pigs. Livestock Science, 2014. 161(0): p. 36-40.

3.Xu, S., J. Lee, and M. Miyake, Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids. Zygote, 2012. 20(02): p. 147-158.

4.Hu, C.H., et al., Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs. Journal of Animal Science, 2013. 91(3): p. 1094-1101.

5.Arce, C., et al., Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium. Comparative Immunology, Microbiology and Infectious Diseases, 2010. 33(2): p. 161-174.

6.Brosnahan, A.J. and D.R. Brown, Porcine IPEC-J2 Intestinal Epithelial Cells in Microbiological Investigations. Veterinary Microbiology, 2011. 156: p. 229-237.

S3Sequencing and blast results of apoptosis relative genes

P53

CTGCTTCCTGAAAACAACCTGCTGTCCTCTGAGCTCTCCCTGGCAGCAGTGAACGATCTGCTGCTGTCCCCAGTCACGAACTGGCTGGATGAAAATCCAGATGACGCCTCCAGAGTGCCAGCGCCTCCTGCAGCAACAGCGCCCGCACCAGCTGCCCCCGCACCAGCCACCTCCTGGCCCCTGTCGTCCTTTGTCCCTTA

Fas

TTGATGCCCAAGTGACTGACCCCGATTCTGAGATGGTGAAACTGAATATTACTAAAAGGGAGTCTGAGTGCCCGGAAGGCCAGCATCGTGAGGGTCAATTCTGCA

Caspase-8

TAGACAAGGGCATCATCATCGGCTCTGATGGGAGGGAAGCCCCCATCTATGATCTGACCTCTTATTTCACTGGTTCGAAGTGCCGTTCCCTTCTTGGTAAACAA

Caspase-9

TAATGCCGATTTGGCTTACGTCCTGAATGCAGACCCCTGTGGCCACTGCCTCATCATCAACAACGTGAATTTCTGCCATGAGTCGGGGCTCAGGACGCGCACTGGCTCCAATATCGACTGTGAGAGGATGCAGCGGCGCTTCCACTTGCTGCGTTTTGTGGTGGACGTGAAGTGCAACCTGACTGCCAAGCAAATGA

Caspase-3

TGTGGGACTGAAGATGACATGGCGTGTCAGAAAATACCAGTTGAGGCAGACTTCTTGTATGCATATTCTACAGCGCCTGGTTACTATTCCTGGCGAAATTCAAAGGACGGATCCTGGTTCATCCAGTCACTTTGTGCAGCGCTGAAACAGTACGCTCACAAGCTTGAGCTTATGCACATTCTTACTCGGGTA

Bcl-2

TACCTGAATGACCACCTAGAGCCTTGGATCCAGGAGAACGGCGGCTGGGACACTTTTGTGGAACTCTACGGAAACAATGCAGCAGCTGAGAGCCGGAAGGGCCAGGAGCGCTTCAACCGATGGTTCCTGACGGGCATGACTCTAGCTGGGGTGGTTCTGCTGGGTTCGCTCTTCAGTCGGA

B2M

TTCACACCGCTCCAGTAGCGATGGCTCCCCTCGTGGCCTTGGTCCTGCTCGGGCTGCTCTCACTGTCTGGCCTGGATGCGGTCGCGCGTCCCCCGAAGGTTCAGGTTTACTCACGCCACCCAGCGGAAAACGGAAAGCCAAATTACCTGAACTGCTATGTATCTGG

β-actin

CCACCACGGCCGAGCGGGAGATCGTGCGGGACATCAAGGAGAAGCTCTGCTACGTCGCCCTGGACTTCGAGCAGGAGATGGCCACCGCCGCGTCCTCCTCCTCCCTGGAGAAGAGCTACGAGCTGCCCA

S4: ANOVA analysis for cell viability in Fig. 2

To make Fig. 2 beautiful, ANOVA data were not showed on the diagram, and now listed in below:

For H2O2 model:

Concentration of H2O2 / Cell viability (%)
(mM) / Mean / SEM / ANOVA analysis*
0 / 99.99737 / 2.24231 / a
0.1 / 96.53283 / 3.72324 / a
0.5 / 64.58794 / 3.34745 / bc
1 / 59.29234 / 2.27043 / c
2 / 45.93477 / 5.03464 / d
3 / 38.84761 / 6.58302 / e
4 / 25.64812 / 6.1933 / f
NAC / 94.94 / 1.79 / a

For X/XO model:

Concentration of XO / Cell viability (%)
(U/L) / Mean / SEM / ANOVA analysis*
0 / 100.00359 / 3.18627 / a
10 / 95.79121 / 2.75705 / a
30 / 69.04711 / 1.62784 / b
50 / 59.24213 / 2.98499 / c
70 / 32.31878 / 5.52296 / d
NAC / 99.95 / 2.27 / a

*Different letters differ significantly(P<0.05, n=3)

S5Pearson correlation coefficient between 21d weaning piglet and H2O2 or X/XO model

Pearson correlation = 0.499, P =0.31

Pearson correlation = 0.946, P =0.04