Fig 1: The cell viability of IPEC-J2 cells treated with active or inactive A. muciniphila. and mRNA level of inflammation related cytokines and tight junctions of the IPEC-J2 cells in different groups. (A) The viability of IPEC-J2 cells co-cultured with living (active) A. muciniphila at 0, 106, 107, 108 and 109 copies/mL for 2.5, 5, 7.5 and 10 h, respectively. (B) The viability of IPEC-J2cells co-cultured with inactive (autoclaved) A. muciniphila at 0, 106, 107, 108 and 109 copies/mL for 2.5, 5, 7.5 and 10 h, respectively. (C) The mRNA level of TNF-a of IPEC-J2 cells in different groups. (D) The mRNA level of IL-1ß of IPEC-J2 cells in different groups. (E) The mRNA level of IL-6 of cells in different groups. (F) The mRNA level of IL-8 of IPEC-J2 cells in different groups. (G) The mRNA level of Occludin of IPEC-J2 cells in different groups. (H) The mRNA level of Occludin of IPEC-J2 cells in different groups. CON, cells treated with PBS for 48 h. TNF, cells challenged with 20 ng/mL Recombinant Susscrofa TNF-a for 48 hours. A, cells co-cultured with 108 copies/mL active A. muciniphila for 48 h. IA, cells co-cultured with109 copies/mL inactive A. muciniphila for 48 h. TNF+A, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 108 copies/mL active A. muciniphila for 7.5 h. TNF+IA, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 109 copies/mL inactive A. muciniphila for 7.5 h. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are expressed as mean ± SEM (n = 3). Results represented one of the three independent experiments.
Fig 2: The apoptosis of IPEC-J2cells in different groups. (A) The profile of flow cytometry with an annexin V-FITC/PI kit of the IPEC-J2 cells in different groups. Each of the frames is divided into four quadrants: Q1, necrotic cells. Q2, cells in the late-stage of apoptosis. Q3, cells in the early-stage of apoptosis. Q4, normal cells. (B) The rate of IPEC-J2 cells in the early stage of apoptosis in different groups. (C) The rate of IPEC-J2 cells in the late stage of apoptosis in different groups. (D) The rate of total apoptosis of IPEC-J2 cells in different groups. Data are represented as mean± SEM (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001. CON, cells treated with PBS for 48 h. TNF, cells challenged with 20 ng/mL Recombinant Susscrofa TNF-a for 48 hours. A, cells co-cultured with 108 copies/mL active A. muciniphila for 48 h. IA, cells co-cultured with 109 copies/mL inactive A. muciniphila for 48 h. TNF+A, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 108 copies/mL active A. muciniphila for 7.5 h. TNF+IA, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 109 copies/mL inactive A. muciniphila for 7.5 h.
Fig 3: Comparison of gene expression abundance and KEGG enrichment in T, and TNF+A and TNF+IA groups. (A) The principal component analysis based on the RNA-Seq data. (B) Venn diagrams of differentially expressed transcripts. (C) The top 20 KEGG pathways enriched in TNF+A group compared to group TNF. (D) The top 20 KEGG pathways enriched in TNF+IA group compared to group TNF. TNF, cells challenged with 20 ng/mL Recombinant Susscrofa TNF-a for 48 hours. TNF+A, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 108 copies/mL active A. muciniphila for 7.5 h. TNF+IA, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 109 copies/mL inactive A. muciniphila for 7.5 h.
Fig 4: The protein-protein interaction network based on the KEGG annotation. (A) Change of protein abundance in TNF+A group compared to group TNF. (B) Change of protein abundance in TNF+IA group compared to group TNF. TNF, cells challenged with 20 ng/mL Recombinant Susscrofa TNF-a for 48 hours. TNF+A, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 108 copies/mL active A. muciniphila for 7.5 h. TNF+IA, cells challenged with 20 ng/mL TNF-a for 40.5 h and then co-cultured with 109 copies/mL inactive A. muciniphila for 7.5 h.
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