Author Contributions
Conceptualization, H.Y. and X.G.; methodology, N.W., M.L. and F.B.; software, K.G.; validation, H.Y. and X.G.; formal analysis, N.W.; investigation, H.Y.; resources, H.Y.; data curation, N.W.; writing—original draft preparation, N.W.; writing—review and editing, N.W.; visualization, N.W., L.M., L.Q. and Y.W.; supervision, H.Y. and X.G; project administration, H.Y.; funding acquisition, H.Y. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Mouse intestinal organoids culture. (A,B) Day 1; (C,D) Day 3; (E) Day 5.
Figure 1.
Mouse intestinal organoids culture. (A,B) Day 1; (C,D) Day 3; (E) Day 5.
Figure 2.
Immunofluorescence of the small intestinal organoids of mice cultured for 10 days. (A–D) Epithelial cells, positive Ep-CAM and E-cadherin staining, (A) stem cells (intestinal stem cells), positive for PCNA staining, (B) Paneth cells, positive for lysozyme, (C) endocrine cells, positive for Chromogranin A staining, (D) clusters of cells (Tuft cells), positive for double cortical protein-like kinase (Doublecortin like kinase, DCLK)-1. Scale base, 50 μM.
Figure 2.
Immunofluorescence of the small intestinal organoids of mice cultured for 10 days. (A–D) Epithelial cells, positive Ep-CAM and E-cadherin staining, (A) stem cells (intestinal stem cells), positive for PCNA staining, (B) Paneth cells, positive for lysozyme, (C) endocrine cells, positive for Chromogranin A staining, (D) clusters of cells (Tuft cells), positive for double cortical protein-like kinase (Doublecortin like kinase, DCLK)-1. Scale base, 50 μM.
Figure 3.
Toxins were added after 14 days of subculture of mouse intestinal organoids. (A–D) Two days after adding OA, the concentrations were 0, 1, 5 and 10 μM, respectively; (E–H) two days after adding CgTx, the concentrations were 0, 0.4, 4 and 20 μM, respectively.
Figure 3.
Toxins were added after 14 days of subculture of mouse intestinal organoids. (A–D) Two days after adding OA, the concentrations were 0, 1, 5 and 10 μM, respectively; (E–H) two days after adding CgTx, the concentrations were 0, 0.4, 4 and 20 μM, respectively.
Figure 4.
Mortality 2 days after OA was added to the mouse intestinal organoids, (A–D) AM/PI photos, the concentrations were 0, 1, 5 and 10 μM, respectively; (E) mortality, one-way ANOVA, p < 0.01, IC50 was 3.611 μM; mortality 2 days after CgTx was added to the mouse intestinal organoids, (F–I) AM/PI photos, the concentrations were 0, 0.4, 4 and 20 μM, respectively; (J) mortality, one-way ANOVA, p < 0.0001, IC 50 was 7.474 μM; * means p < 0.05, **means p < 0.01, **** means p < 0.0001.
Figure 4.
Mortality 2 days after OA was added to the mouse intestinal organoids, (A–D) AM/PI photos, the concentrations were 0, 1, 5 and 10 μM, respectively; (E) mortality, one-way ANOVA, p < 0.01, IC50 was 3.611 μM; mortality 2 days after CgTx was added to the mouse intestinal organoids, (F–I) AM/PI photos, the concentrations were 0, 0.4, 4 and 20 μM, respectively; (J) mortality, one-way ANOVA, p < 0.0001, IC 50 was 7.474 μM; * means p < 0.05, **means p < 0.01, **** means p < 0.0001.
Figure 5.
ATPase assay of 2 days after added (A) OA; (B) CgTx of mouse intestinal organoids via CellTiter-Glo® (Promega), one-way ANOVA, (A,B) p < 0.0001; ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001.
Figure 5.
ATPase assay of 2 days after added (A) OA; (B) CgTx of mouse intestinal organoids via CellTiter-Glo® (Promega), one-way ANOVA, (A,B) p < 0.0001; ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001.
Figure 6.
TUNEL assay of 2 days after OA was added to the mouse intestinal organoids, (A–D) TUNEL photos, the concentrations were 0, 1, 5 and 10 μM, respectively; (E) count of apoptosis cells, one-way ANOVA, p > 0.05; TUNEL assay of 2 days after CgTx was added to the mouse intestinal organoids, (F–I) TUNEL photos, the concentrations were 0, 0.4, 4 and 20 μM, respectively; (J) count of apoptosis cells, the fluorescence images obtained were not of adequate resolution to include, one-way ANOVA, p > 0.05.
Figure 6.
TUNEL assay of 2 days after OA was added to the mouse intestinal organoids, (A–D) TUNEL photos, the concentrations were 0, 1, 5 and 10 μM, respectively; (E) count of apoptosis cells, one-way ANOVA, p > 0.05; TUNEL assay of 2 days after CgTx was added to the mouse intestinal organoids, (F–I) TUNEL photos, the concentrations were 0, 0.4, 4 and 20 μM, respectively; (J) count of apoptosis cells, the fluorescence images obtained were not of adequate resolution to include, one-way ANOVA, p > 0.05.
Figure 7.
Heatmap of after 14 days of subculture of mouse intestinal organoids, different mRNA values corresponding to the box map of all gene expression values (FPKM) were selected for the clustering of heatmap visualization, (A) added with OA for 2 days, and the concentration was 5 μM; (B) added with CgTx for 2 days, and the concentration was 4 μM.
Figure 7.
Heatmap of after 14 days of subculture of mouse intestinal organoids, different mRNA values corresponding to the box map of all gene expression values (FPKM) were selected for the clustering of heatmap visualization, (A) added with OA for 2 days, and the concentration was 5 μM; (B) added with CgTx for 2 days, and the concentration was 4 μM.
Table 1.
Differential gene expression analysis of RNA-seq of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
Table 1.
Differential gene expression analysis of RNA-seq of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
UpDown | Type | GeneSymbol |
---|
DOWN | protein_coding | Ngfr |
DOWN | protein_coding | Ifrd1 |
DOWN | protein_coding | Ccne1 |
DOWN | protein_coding | Peg3 |
DOWN | protein_coding | Klf4 |
DOWN | protein_coding | Slbp |
DOWN | protein_coding | Hmox1 |
DOWN | protein_coding | Trim28 |
DOWN | protein_coding | Adamts4 |
DOWN | protein_coding | Tgfbr1 |
DOWN | protein_coding | Hnrnph1 |
DOWN | protein_coding | Carhsp1 |
DOWN | protein_coding | Slc3a2 |
DOWN | protein_coding | Zfp296 |
DOWN | protein_coding | Dnajb9 |
DOWN | protein_coding | Gadd45b |
DOWN | protein_coding | Sqstm1 |
DOWN | protein_coding | Plagl1 |
DOWN | protein_coding | Tnfaip3 |
DOWN | protein_coding | Sirt1 |
Table 2.
Differential gene expression analysis of RNA-seq of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
Table 2.
Differential gene expression analysis of RNA-seq of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
UpDown | Type | GeneSymbol |
---|
DOWN | protein_coding | Peg3 |
DOWN | protein_coding | Zim1 |
UP | protein_coding | Fosb |
UP | protein_coding | Cdc20 |
UP | protein_coding | Sulf2 |
DOWN | protein_coding | Id3 |
DOWN | protein_coding | Slc16a12 |
DOWN | protein_coding | B4galnt2 |
DOWN | protein_coding | 2310057J18Rik |
DOWN | protein_coding | Tmprss6 |
DOWN | protein_coding | Gabrp |
DOWN | protein_coding | Cpm |
DOWN | protein_coding | Id2 |
UP | protein_coding | Olfm4 |
UP | protein_coding | Tgm1 |
UP | protein_coding | Nr4a1 |
DOWN | protein_coding | Sult1c2 |
DOWN | protein_coding | Cldn6 |
DOWN | protein_coding | Cacna1h |
UP | protein_coding | Il33 |
Table 3.
GO of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
Table 3.
GO of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
Ontology | Description |
---|
CC | mitochondrial inner membrane |
CC | organelle inner membrane |
CC | mitochondrial protein-containing complex |
BP | mitochondrion organization |
CC | Golgi apparatus subcompartment |
CC | mitochondrial matrix |
BP | organophosphate biosynthetic process |
BP | nucleoside phosphate metabolic process |
BP | nucleotide metabolic process |
BP | generation of precursor metabolites and energy |
Table 4.
GO of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
Table 4.
GO of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
Ontology | Description |
---|
MF | receptor ligand activity |
CC | anchored component of membrane |
BP | organic acid biosynthetic process |
BP | wound healing |
CC | collagen-containing extracellular matrix |
BP | carboxylic acid biosynthetic process |
BP | cell-substrate adhesion |
BP | leukocyte migration |
BP | cell chemotaxis |
BP | negative regulation of locomotion |
Table 5.
KEGG analysis of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
Table 5.
KEGG analysis of mouse intestinal organoids were subcultured for 14 days and added OA for 2 days, concentration 5 μM.
Description |
---|
Chemical carcinogenesis–reactive oxygen species |
Prion disease |
Peroxisome |
Pathways of neurodegeneration–multiple diseases |
Thermogenesis |
Alzheimer disease |
Salmonella infection |
Parkinson disease |
Oxidative phosphorylation |
TNF signaling pathway |
Table 6.
KEGG analysis of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
Table 6.
KEGG analysis of mouse intestinal organoids were subcultured for 14 days and added CgTx for 2 days, concentration 4 μM.
Description |
---|
Cytokine-cytokine receptor interaction |
Calcium signaling pathway |
Cholesterol metabolism |
Arachidonic acid metabolism |
Mineral absorption |
Hippo signaling pathway |
Axon guidance |
PPAR signaling pathway |
Gastric acid secretion |
Sulfur metabolism |