Journal: PLOS One

Article Title: Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

doi: 10.1371/journal.pone.0359086

Figure Lengend Snippet: (A) Porcine small intestinal crypts were embedded in Matrigel domes and cultured to establish small intestinal organoids. (B) Mature organoids were treated with 20 μM lipopolysaccharide (LPS) for 24 h to induce inflammation. (C) Treated organoids were analyzed using qRT-PCR, DAPI and PI staining, and immunofluorescence to assess gene expression, cell viability, and protein expression.

Article Snippet: The stained organoids were washed with PBS, mounted on glass slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and nuclear staining was performed simultaneously.

Techniques: Cell Culture, Organoids, Quantitative RT-PCR, Staining, Immunofluorescence, Gene Expression, Expressing

Journal: PLOS One

Article Title: Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

doi: 10.1371/journal.pone.0359086

Figure Lengend Snippet: (A) Phase-contrast images of pSIOs treated with LPS (0, 20, and 30 μM) showing structural disruption, core darkening, and fragmentation at higher concentrations compared with controls. Scale bars: 200 μm. (B) DAPI (blue) and PI (red) staining of pSIOs treated with LPS (5, 10, 20, and 30 μM) showing a dose-dependent increase in apoptosis. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each LPS concentration in each biological replicate. Scale bars: 200 μm. (C) Representative immunofluorescence images of E-cadherin (green) and DAPI (blue) staining of pSIOs showing reduced and discontinuous E-cadherin expression in LPS-treated organoids compared with controls. Enlarged views of the yellow boxed regions are shown. Low-magnification (200 μm) images and enlarged views (50 μm) are shown in 3C. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. (D) Relative gene expression levels of CDH1 and TJP1 in pSIOs treated with LPS (20 μM) compared with controls (0 μM), as determined by qRT-PCR. LPS treatment significantly decreased the expression of CDH1 and TJP1 , indicating disruption of epithelial barrier integrity. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. *** p < 0.001.

Article Snippet: The stained organoids were washed with PBS, mounted on glass slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and nuclear staining was performed simultaneously.

Techniques: Phase Contrast, Disruption, Staining, Organoids, Concentration Assay, Immunofluorescence, Expressing, Gene Expression, Quantitative RT-PCR

Journal: PLOS One

Article Title: Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

doi: 10.1371/journal.pone.0359086

Figure Lengend Snippet: (A) Immunofluorescence staining of TLR4 in pSIOs after LPS (20 μM) treatment. Nuclei were stained with DAPI (blue), and TLR4 expression is shown in green. Representative images suggest higher TLR4 immunofluorescence in the LPS-treated group than in the control group. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. Scale bar = 100 μm. (B) qRT-PCR analysis of pro- and anti-inflammatory cytokines in pSIOs after LPS (20 μM) exposure. Pro-inflammatory cytokines TNF-α , IL-1β , and IL-8 were significantly upregulated in the LPS-treated group compared with the control group. Anti-inflammatory cytokines IL-4 and IL-10 also showed significantly increased expression, whereas TGF-β expression was increased but did not reach statistical significance following LPS stimulation. Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. * p < 0.05, ** p < 0.01.

Article Snippet: The stained organoids were washed with PBS, mounted on glass slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and nuclear staining was performed simultaneously.

Techniques: Immunofluorescence, Staining, Expressing, Control, Organoids, Quantitative RT-PCR, Analysis

Journal: PLOS One

Article Title: Lipopolysaccharide-induced alterations in barrier integrity and inflammatory signaling in porcine small intestinal organoids

doi: 10.1371/journal.pone.0359086

Figure Lengend Snippet: (A) Immunofluorescence of pSIOs stained with DAPI (blue) to visualize nuclei and immunolabeled for SGLT1 (green), PEPT1 (green), GLUT2 (green), TGR5 (red), and GLP-1 (green). Representative untreated (upper rows) and LPS-treated (lower rows) organoids are shown. Untreated organoids exhibited distinct localization of SGLT1, PEPT1, GLUT2, and TGR5 along the epithelial membrane with strong expression and cytoplasmic GLP-1 expression in scattered enteroendocrine-like cells within the epithelial layer. LPS-treated organoids exhibited reduced expression intensity, irregular protein distribution, and partial loss of membrane localization. Enlarged views of the yellow boxed regions further highlight differences in expression patterns and subcellular localization between untreated and LPS-treated organoids. Images are representative of three independent biological replicates (n = 3), with at least five organoids analyzed for each treatment condition in each biological replicate. Scale bars: 50 μm. (B) qRT-PCR analysis showing significant alterations in nutrient transporter and enteroendocrine-related gene expression following LPS treatment. Decreased SLC5A1 (SGLT1) and SLC15A1 (PEPT1) expression and increased SLC2A2 expression were observed after LPS exposure. Among enteroendocrine-related genes, GPBAR1 expression was markedly decreased, whereas GCG (the precursor gene of GLP-1) expression was significantly increased after LPS exposure.Data are presented as the mean ± SD from three biological replicates (n = 3); each biological replicate was analyzed using five technical replicates. ** p < 0.01, *** p < 0.001.

Article Snippet: The stained organoids were washed with PBS, mounted on glass slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and nuclear staining was performed simultaneously.

Techniques: Immunofluorescence, Staining, Immunolabeling, Organoids, Membrane, Expressing, Quantitative RT-PCR, Analysis, Gene Expression