Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Toxicity of ethanol extracted of dried E. cava (EEEC), eckol, dieckol, and phlorofucofuroeckol ( A ) EEEC, eckol, dieckol, and phlorofucofuroeckol toxicity was evaluated by performing a cell viability assay using IPEC-J2 cells incubated with the indicated concentrations of each compound for 24 h. ( B ) Heparin-binding epidermal growth factor-like growth factor ( HBEGF) expression in the treatment groups (EEEC, eckol, dieckol, and phlorofucofuroeckol). In qRT-PCR analysis, target gene expression levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). Error bars indicate standard error of triplicate analyses. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Viability Assay, Incubation, Binding Assay, Expressing, Quantitative RT-PCR, Targeted Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Eckol induces PDX1 and HBEGF expression. PDX1 mRNA ( A ) and protein ( B ) expression in cells treated with eckol at different concentrations (0 µM, 10 µM, 20 µM, 50 µM, 100 µM, and 200 µM). HBEGF mRNA ( C ) and protein ( D ) expression in cells treated with various concentrations of eckol. Protein band intensity in the Western blot analysis was quantified using the threshold function of the ImageJ software. For qRT-PCR analysis, target gene expression levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). Error bars indicate standard error of triplicate analyses. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Expressing, Western Blot, Software, Quantitative RT-PCR, Targeted Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Eckol-induced PDX1 regulates HBEGF expression. ( A ) Activity of HBEGF upstream region. IPEC-J2 cells were transfected with DNA sequences of different lengths (−2000, −1500, −1000, and −500) comprising the upstream region of HBEGF (n = 3). ( B ) Upstream sequence of the putative core region of the HBEGF promoter. Sequence numbering is relative to the transcription start site. Putative PDX1 binding sites (TAAT) are boxed and labeled above (upstream of −954, −790, and −767). ( C ) Deletion assay of putative PDX1 binding sites. Sequences in which the deleted binding sites (upstream of −954, −790, and −767) were transfected into IPEC-J2 cells (n = 3). ( D ) PDX1 knockdown assay. The siRNA-mediated suppression of PDX1 in IPEC-J2 cells was confirmed using qRT-PCR. ( E ) Relative HBEGF expression in PDX1 -silenced IPEC-J2 cells treated with eckol. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase, and the relative expression level was normalized to that of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). Error bars indicate standard error of triplicate analyses. * p < 0.05. Lower-case letters indicate significant differences among treatments based on Duncan’s multiple range test.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Expressing, Activity Assay, Transfection, Sequencing, Binding Assay, Labeling, DNA Deletion Assay, Knockdown, Quantitative RT-PCR, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Effects of eckol on intestinal barrier function. Activation of PI3K/AKT ( A ) and P38 ( B ) signaling pathways in IPEC-J2 cells treated with eckol and HBEGF for 2 h under lipopolysaccharide (LPS) challenge. AKT (Ser473) and P38 (Thr180/Tyr182) phosphorylation was evaluated by Western blotting. Protective effects of eckol and HBEGF on intestinal barrier function in IPEC-J2 cells under LPS challenge, as determined by TEER quantification ( C ), and the permeability of fluorescein isothiocyanate-FD-4 ( D ) (n = 3). ZO-1 expression upon eckol and HBEGF treatment under LPS challenge was evaluated by immunofluorescence analysis ( E ) and Western blotting ( F ). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). IPEC-J2 cells were treated with eckol and HBEGF under LPS challenge with or without PI3K/AKT (LY2944002) and P38 (SB202190) inhibitors. Error bars indicate standard error of triplicate analyses. * p < 0.05, ** p < 0.01, *** p < 0.001. Lower-case letters indicate significant differences among treatments that are based on Duncan’s multiple range test.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Western Blot, Permeability, Expressing, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Eckol promotes intestinal wound healing through HBEGF. ( A ) Cell proliferation was assessed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h using the water-soluble tetrazolium-1 assay (n = 3) ( B ) Migration was assessed at 0 h, 6 h, and 12 h using a wound-healing assay (n = 3) ( C ) Relative expression levels of cell migration-related genes were analyzed using qRT-PCR in eckol- and HBEGF-treated IPEC-J2 cells under LPS challenge with or without PI3K/AKT (LY2944002) and P38 (SB202190) inhibitors. Error bars indicate standard error of triplicate analyses. Lower-case letters indicate significant differences ( p < 0.05) among treatments, based on Duncan’s multiple range test.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Migration, Wound Healing Assay, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: List of primers.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Sequencing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF

doi: 10.3390/ijms21134755

Figure Lengend Snippet: Schematic illustration of the effects of eckol on intestinal development during the suckling-to-weaning transition. Eckol induces PDX1 expression that directly regulates HBEGF expression, leading to enhanced intestinal barrier function, accelerated wound healing, and reduced oxidative stress through the activation of various intracellular signaling pathways.

Article Snippet: The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.

Techniques: Expressing, Activation Assay, Protein-Protein interactions