Journal: Molecules

Article Title: The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

doi: 10.3390/molecules26247495

Figure Lengend Snippet: In silico analysis 3D results of the possible covalent interactions between the target TLR4/NF-κB proteins and the target molecules including galangin ( a,c ), quercetin ( b,d ), and the inhibitor BAY 11-7082 ( e ).

Article Snippet: The primary antibodies used for phosphor-NF-κB p65 (p-p65) (Bioss bs-0982R), TLR4 (Bioss bs-20594R), and β-actin (Bioss bs-0061R) were all provided by Biosynthesis Biotechnology Inc. (Beijing, China).

Techniques: In Silico

Journal: Molecules

Article Title: The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

doi: 10.3390/molecules26247495

Figure Lengend Snippet: Analysis results for the affinity of two flavonols or inhibitor (BAY 11-7082) to TLR4 or NF-κB.

Article Snippet: The primary antibodies used for phosphor-NF-κB p65 (p-p65) (Bioss bs-0982R), TLR4 (Bioss bs-20594R), and β-actin (Bioss bs-0061R) were all provided by Biosynthesis Biotechnology Inc. (Beijing, China).

Techniques: Binding Assay

Journal: Molecules

Article Title: The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

doi: 10.3390/molecules26247495

Figure Lengend Snippet: The 2D results for the chemical groups involved in the interactions between TLR4/NF-κB proteins and the target molecules including galangin ( a,c ), quercetin ( b, d ), and the inhibitor BAY 11-7082 ( e ).

Article Snippet: The primary antibodies used for phosphor-NF-κB p65 (p-p65) (Bioss bs-0982R), TLR4 (Bioss bs-20594R), and β-actin (Bioss bs-0061R) were all provided by Biosynthesis Biotechnology Inc. (Beijing, China).

Techniques:

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 6. The Expression of TLR4 protein in November was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are shown at low magnification in A, E, I, and M, respectively; B, F, J, and n refer to the amplification of the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in magnum, isthmus, uterus, and vagina are described in C, G, K, and o; respectively; and D, H, I, and P represent the negative controls for the magnum, isthmus, uterus, and vagina, respectively. No immunoreaction products were observed. All sections were counterstained with hematoxylin. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), blood vessel endothelium (black thin arrow head). Scale bars: 100 lm (E and M), 50 lm (A, D, H, I, L, and P), and 10 lm (B, C, F, G, J, K, N, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Expressing, Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 7. The Location of TLR4 protein was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are depicted in the pictures (A, E, I, and M) at low magnification: B, F, J, and n refer to the amplification for the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in the magnum, isthmus, uterus, and vagina are described C, G, K, and O, respectively; and D, H, I, and p show the negative controls for the magnum, isthmus, uterus, and vagina, respectively. All sections were counterstained with hematoxylin. No immunoreaction products were observed. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), and blood vessel endothelium (black thin arrow head). Scale bars: 50 lm (A, D, E, H, I, L, M, and P), 20 lm (B, F, J, and N), and 10 lm (C, G, K, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 8. The IOD of TLR2/4 Positive Reaction Parts in Oviduct during Hibernation. (A) Image shows changes in numbers of TLR2-positive reaction parts in four different parts of oviduct in two different months. (B) The IOD of TLR4- positive reaction parts in oviduct during hibernation. Data are presented as mean SE of five turtles per group. Significant differences are identified as *, P < 0.05; **, P < 0.01.

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques:

Journal: Pharmaceutical Biology

Article Title: Berberine attenuates septic cardiomyopathy by inhibiting TLR4/NF-κB signalling in rats

doi: 10.1080/13880209.2021.1877736

Figure Lengend Snippet: Effects of berberine on key effectors of the TLR4/NF-κB signalling pathway. (A) TLR4 protein expression in the myocardial tissue and quantitation. (B) Nuclear p65 protein expression in the rat sepsis cardiomyopathy model measured by ELISA. Effects of berberine on TNFα (C) and IL 1β (D) levels in the myocardial tissue. * p < 0.001 indicates significance in comparison with the Con group. △ p < 0.001 indicates significance in comparison with the LPS group.

Article Snippet: The membranes were blocked with 5% skim milk, followed by successive incubations with rabbit anti-mouse TLR4 polyclonal antibody (1:2000, Santa Cruz, USA) at 4 °C overnight and mouse anti-rabbit IgG monoclonal secondary antibody (1:10000, Bioss, China) for 2 h. The membranes were next incubated with ECL ultra western HRP substrate (Millipore, Billerica, MA, USA) and detected on a gel imaging system (ImageQuant LAS 4000, GE Healthcare, USA).

Techniques: Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: Generation of hsa-miR-24-3p is dependent on CR3 and TLR4. (a) The level of hsa-miR-24-3p 1 h after infection was higher in MEV Ca than in MEV. Blocking of CR3 or TLR4 on blood monocytes had a minor effect on hsa-miR-24-3p content in vesicles. However, when both CR3 and TLR4 were blocked, hsa-miR-24-3p was absent from vesicles. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. EVs were isolated from 10 6 human monocytes, and hsa-miR-24-3p levels were determined using qPCR. (b) Mmu-miR-24-3p content was significantly higher in REV Ca (vesicles from opsonized C. albicans -induced RAW 264.7 cells) than in REV (vesicles from untreated RAW 264.7 cells). The level of mmu-miR-24-3p was not elevated in CR3- or TLR4 -silenced RAW 264.7 cells. Data represent mean values ± SD, P = 0.0245 and P = 0.0166, unpaired two-tailed t test, n = 3 or 4 different experiments. (c) Candida -treated CD11b- and TLR4 -silenced RAW 264.7 cells generated the same amount of REV as nonsilenced cells. EVs isolated from the same numbers of cells were counted by DLSM. (d) CD11b and TLR4 colocalized on blood monocytes upon incubation with soluble β-glucan (sβG) and mannan but not on untreated cells, as observed by CLSM. Green, CD11b; red, TLR4. Bars, 10 μm. Data are representative of n = 3 independent experiments. (e) Colocalization of CD11b and TLR4 in the presence of soluble β-glucan (sβG) and mannan on monocytes, as confirmed by PLA. Red, CD11b–TLR4 complexes; blue, DNA. Bars, 10 μm. Data are representative of n = 3 experiments. (f) EVs isolated from sβG and mannan-treated human monocytes for 1 h (MEV sβG+mannan ) contained significantly more hsa-miR-24-3p than MEV. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. REV Ca but not REV Ca(TLR4 and CD11b silenced) induced significant growth (g) and hyphal filamentation (h) in C. albicans . EVs were isolated from opsonized C. albicans -infected or control RAW 264.7 cells (REV and REV Ca , respectively). EVs were also isolated from opsonized C. albicans - infected or control TLR4- and CD11b-silenced RAW 264.7 cells [REV (TLR4 and CD11b silenced) and REV Ca(TLR4 and CD11b silenced) , respectively]. Isolated EVs were counted by DLSM, and C. albicans was incubated with identical amounts of vesicles obtained from the indicated treatments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Infection, Blocking Assay, Two Tailed Test, Isolation, Generated, Incubation

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: hsa-miRNA-24-3p acts across species boundaries (cross-kingdom) as it regulates C. albicans gene expression. C. albicans induces the release of miRNA-containing vesicles in human monocytes upon binding of mannan and soluble β glucan to TLR4 and CR3, respectively, followed by receptor colocalization. Subsequently, EVs are released from multivesicular bodies transporting miRNAs. EVs attach via CR1 on the vesicle to opsonized C. albicans . hsa-miRNA-24-3p but not hsa-miRNA-21-5p inhibits sol1 translation in C. albicans , leading to enhanced growth and filamentation of the fungus. Graphic created with BioRender.com.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Expressing, Binding Assay