Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: ( A ) Effect of BB1 on TLR-2/TLR-6 or TLR-2/TLR-1 heterodimer binding and signal-transduction pathway activation as determined by SEAP (secreted embryonic alkaline phosphatase) activity in TLR-1 or TLR-6 knock-out HEK-293 reporter cell lines (InvivoGen, San Diego, CA). BB1 produced a marked increase in SEAP activity in TLR-2/TLR-6 (TLR-1 -/- ), but not TLR-2/TLR-1 (TLR-6 -/- ), expressing HEK-293 cells. PAM3cBK4 (positive control) produced an increase in SEAP activity in TLR-2/TLR-1 (TLR-6 -/- ) HEK-293 cells. The siRNA knock-down of TLR-1 or TLR-6, but not TLR-2, inhibited the BB1-induced increase in occludin mRNA ( B ) and protein and densitometry analysis ( C, D ) and Caco-2 TEER ( E ) ∗∗∗∗ P < 0.0001 versus control, NS (not significant).

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Binding Assay, Transduction, Activation Assay, Activity Assay, Knock-Out, Produced, Expressing, Positive Control, Knockdown, Control

Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: A BB1, but not BB4, treatment produced a focal aggregation of TLR-2 at the Caco-2 apical membrane surface and translocation of TOLLIP towards the apical membrane surface from the medial and basal regions of Caco-2 cells, as assessed by immunostaining. (TOLLIP, green; TLR-2 red; and DAPI blue). Scale bars = 10 μm. Quantification of TOLLIP apical translocation immunostaining intensity using ImageJ. ∗∗∗∗ P < 0.0001 versus control, NS (not significant) ( B ). The siRNA knockdown of TLR-2 or IRAK-1 inhibited the BB1-induced apical translocation of TOLLIP. The siRNA-induced of TOLLIP inhibited the BB1-induced increase in occludin mRNA ( C ) and protein expression ( D ), densitometry ( E ) and Caco-2 TEER ( F ). ∗∗∗∗ P < 0.0001 versus control, NS (not significant).

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Produced, Membrane, Translocation Assay, Immunostaining, Control, Knockdown, Expressing

Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: A BB1, but not BB4, caused an increase in IRAK-1 phosphorylation (Thr209) as assessed by immunoblotting. The siRNA-induced knockdown of IRAK-1 inhibited the BB1-induced increase in occludin mRNA ( B ), occludin protein ( C ), densitometry ( D ) and Caco-2 TEER ( E ) (means ± SE, n = 4). ∗∗∗∗ P < 0.0001 versus control, NS (not significant). The siRNA-induced knockdown of TLR-2 or TLR-6, but not TLR-1, inhibited the BB1 increase in IRAK-1 phosphorylation, but had no effect on total IRAK-1 protein expression ( F ).

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Control, Expressing

Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: A Oral -gastric gavage of BB1 [1 × 10 9 colony-forming units (CFUs)/mL], but not BB4, produced a decrease in mouse small intestinal permeability to dextran 10 kDa. P **** < 0.0001 versus control. BB1 administration caused an increase in occludin mRNA ( B ) and protein ( C ), densitometry ( D ) in the mouse intestinal tissue (24-hour treatment period). E BB1 administration also produced an increase in occludin expression and localization at the apical membrane surface and junctional areas, as assess by immunostaining. (Occludin, green; nuclei, blue). Scale bars = 10 μm ∗ P < 0.05, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001, NS (not significant). ( E ). BB1 oral-gastric gavage administration produced a cytoplasmic-to-apical membrane translocation of TOLLIP in mouse intestinal epithelial cells ( F ). The BB1-induced apical membrane TOLLIP recruitment was inhibited in the TLR-2 ΔIEC mice (TOLLIP, green; occludin, red; (Leica SP8 confocal microscope).

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Produced, Permeability, Control, Expressing, Membrane, Immunostaining, Translocation Assay, Microscopy

Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: A BB1 oral-gastric gavage produced an increase in small intestinal tissue expressions of TLR-2 and TLR-6 but not TLR-1. BB1 also produced an increase in apical membrane expression and localization of TLR-2 in mouse intestinal epithelial cells in the wild-type (WT) mice but not in the TLR-2 ΔIEC mice, as assessed by immunostaining (TLR-2, green; occludin, red; nuclei, blue; magnification 63x) ( B ). The BB1-induced increase in mouse intestinal tissue occludin mRNA ( C ) and protein ( D ), densitometry ( E ) and apical membrane and junctional localization of occludin (occludin, green; nuclei, blue) ( F ), and decrease in intestinal permeability to dextran 10 kDa ( G ) were inhibited in the TLR-2 ΔIEC mice. ∗∗∗∗ P < 0.0001 versus control, NS (not significant).

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Produced, Membrane, Expressing, Immunostaining, Permeability, Control

Journal: NPJ Biofilms and Microbiomes

Article Title: Probiotic bacteria Bifidobacterium bifidum upregulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 receptor complex activation of occludin gene

doi: 10.1038/s41522-025-00903-7

Figure Lengend Snippet: BB1 attaches to the apical membrane surface of the host intestinal epithelial cells by binding to the TLR-2/TLR-6 heterodimer complex and induces the phosphorylation of IRAK-1 and apical translocation of TOLLIP, which, in turn, leads to the down-steam activation of occludin gene and occludin protein synthesis and occludin-dependent upregulation of the intestinal TJ barrier.

Article Snippet: Samples were incubated with anti-occludin antibody, anti-TOLLIP, or TLR-2 antibody (Cell Signaling Technology; Danvers, MA) overnight.

Techniques: Membrane, Binding Assay, Phospho-proteomics, Translocation Assay, Serial Time-encoded Amplified Microscopy, Activation Assay

Journal: Current Issues in Molecular Biology

Article Title: Protective Effects of Magnolia kobus DC. Extract on Inflammatory Response and Alveolar Bone Loss in Ligature-Induced Periodontitis Rats

doi: 10.3390/cimb48010109

Figure Lengend Snippet: Inhibitory effect of MKE on the TLR4-NFκB signaling pathway. The expression levels of TLR-4, p-IκBα/IκBα, and p-p65/p65 in tissues collected at week 16 were evaluated. ( A ) Representative Western blot image. ( B ) Quantitative analysis of TLR-4 expression. ( C ) Quantitative analysis of the p-IκBα/IκBα expression ratio. ( D ) Quantitative analysis of the p-p65/p65 ratio. The data are expressed as mean ± standard deviation ( n = 3/group). * p < 0.05, ** p < 0.01 compared to the ligature control group; # p < 0.05 versus the non-ligature control group. Non-ligature = non-ligature control group, Ligature = ligature control group, Doxycycline = ligature + doxycycline 20 mg/kg group, MKE 100 = ligature + MKE 100 mg/kg group, MKE 400 = ligature + MKE 400 mg/kg group.

Article Snippet: Toll-like receptor 4 (TLR-4) , Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Current Issues in Molecular Biology

Article Title: Protective Effects of Magnolia kobus DC. Extract on Inflammatory Response and Alveolar Bone Loss in Ligature-Induced Periodontitis Rats

doi: 10.3390/cimb48010109

Figure Lengend Snippet: The postulated mechanism underlying the anti-periodontitis activity of MKE. MKE = Magnolia kobus DC. extract, ROS = reactive oxygen species, TLR-4 = toll-like receptor 4, p-IκBα = phosphorylated inhibitor of κBα, IκBα = inhibitor of κBα, IL-1β = interleukin-1β, IL-6 = interleukin-6, TNF-α = tumor necrosis factor-α, COX-2 = cyclooxygenase-2, MMPs = matrix metalloproteinases.

Article Snippet: Toll-like receptor 4 (TLR-4) , Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay