Journal: Scientific reports

Article Title: Myricetin ameliorates ox-LDL-induced HUVECs apoptosis and inflammation via lncRNA GAS5 upregulating the expression of miR-29a-3p.

doi: 10.1038/s41598-021-98916-7

Figure Lengend Snippet: Figure 7. Myr inactivated the TLR4/NF-κB signalling pathway in ox-LDL-treated HUVEC by down- regulating GAS5 or upregulating miR-29a-3p. Western blot analysis of p-p65, p65T, p-IκBα, IκBα and TLR4. *P < 0.05 compared with Control; #P < 0.05 compared with ox-LDL + Myr + NC. &P < 0.05 compared with ox-LDL + Myr + mimic control.

Article Snippet: Protein samples isolated from HUVEC cells (25 μg)) were resolved using SDS-PAGE and then moved to polyvinylidene fluoride (PVDF) membrane (Millipore, USA) with being probed with GAPDH (#2118, 1:5000, Cell Signaling), Bax (#2772S, 1:1000, Cell Signaling), Bcl-2 (#15071S, 1:1000, Cell Signaling), caspase3 (#9662S, 1:1000, Cell Signaling), CD31 (ab9498, 1:1000, Abcam), SM22a (ab14106, 1:1000, Abcam), p-p65 (10745-1-AP, 1:1000, Proteintech), p65 (66535-1-Ig, 1:1000, Proteintech), p-IkBa (10268-1-AP, 1:1000, Proteintech), IkBa (66418-1-Ig, 1:1000, Proteintech) and TLR4 (66350-1-Ig, 1:1000, Proteintech) antibody followed by appropriate secondary antibody.

Techniques: Western Blot, Control

Journal:

Article Title: Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2

doi: 10.1128/IAI.69.12.7387-7395.2001

Figure Lengend Snippet: CD14, TLR2, and TLR4 mRNA expression in HGEC. (A) Expression of human CD14, TLR2, and TLR4 mRNA was analyzed by RT-PCR as detailed in Materials and Methods. Human monocytes were used as positive sources of CD14, TLR2, and TLR4 mRNA expression to confirm the specificity of the primers and PCR. The β-actin gene was assayed as a positive control. PCR products of non-RT samples were examined as a negative control. Lane M, Size markers. (B) Ethidium bromide-stained PCR products were photographed, and then the images were digitized and analyzed. Results are expressed as the ratio of each PCR product to β-actin band density. Data represent three independent experiments. PCR was performed in duplicate for each assay.

Article Snippet: For TLR4 detection, the cells were stained with goat polyclonal antibody to human TLR4 (Santa Cruz Biotech., Inc., Santa Cruz, Calif.), followed by FITC-conjugated rabbit anti-goat IgG (Zymed Lab., Inc.).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Staining

Journal:

Article Title: Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2

doi: 10.1128/IAI.69.12.7387-7395.2001

Figure Lengend Snippet: CD14, TLR2, and TLR4 expression in HGEC. Expression of CD14, TLR2, and TLR4 in HGEC was determined by staining with specific antibodies (bold line) or their isotype as a control (thin line) as detailed in Materials and Methods. Human monocytes were used as positive sources of CD14, TLR2, and TLR4.

Article Snippet: For TLR4 detection, the cells were stained with goat polyclonal antibody to human TLR4 (Santa Cruz Biotech., Inc., Santa Cruz, Calif.), followed by FITC-conjugated rabbit anti-goat IgG (Zymed Lab., Inc.).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Systematic development of peptide inhibitors targeting the CXCL12/HMGB1 interaction

doi: 10.1101/2019.12.18.878504

Figure Lengend Snippet: HMGB1-induced release of IL6 and TNF via TLR4 is not inhibited by HBP08. The concentration of IL6 ( A ) and TNF ( B ) in the supernatant of monocytes treated with HMGB1 or LPS in the presence of HBP08 or a neutralizing antibody against TLR4 was measured by CBA. Data are shown as mean±SEM of at least four independent experiments performed. ***p<0.001; ****p<0.0001 by one-way ANOVA followed by Dunnett’s multicomparisons test.

Article Snippet: A polyclonal neutralizing antibody against TLR4 (AF1478, R&D System,) was used to block TLR4 engagement.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: TLR2 and TLR4 expression in the colonia mucosa of control patients according to the gender and age.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: ( A ) Correlation between TLR2 and TLR4 mRNA expression in the whole group of IBS patients (r s = 0.78, p<0.0001). ( B ) Changes in mRNA expression levels of TLR2 and ( C ) TLR4 in controls and in patients with predominant diarrhea (IBS-D), or constipation (IBS-C) or mixed (IBS-M) assessed by real-time PCR. Values are expressed as mean ± SEM. * Represents p<0.05 using Mann-Whitney test.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: Correlation between TLR2 ( A ) and TLR4 ( B ) expressions in the colonic mucosa and the duration of symptoms of IBS patients (n = 46, r s = 0.34, p = 0.02 for TLR2 and r s = 0.37, p = 0.01 for TLR4). Data were correlated by using non-parametric Spearman test.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

Techniques:

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: Representative photomicrographs show the distribution of TLR2 and TLR4 proteins in the colonic mucosa of controls and IBS patients according to the disease subtype (IBS-Constipated, IBS-Diarrhea or IBS-Mixed alternating constipation with diarrhea) Red, TLR staining; blue, DAPI nuclear staining. Original magnification, ×20.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

Techniques: Staining

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: Epithelials cells obtained from the colonic mucosa were assessed by flow cytometry as described in Patients and Methods. Mucosal colonic cells were double-stained with isotype IgG control or mAbs to TLR2, TLR4 or CD14 together with APC-conjugated anti Epcam mAbs. ( A ) Representative histogram showing the fluorescence intensity of surface TLR4 expression in gated Epcam+ cells from all IBS subtypes and controls patients. Changes in TLR4 surface expression ( B ) , intracellular TLR4 expression ( C ) , CD14 expression ( D ) and TLR2 surface expression ( E ) in IECs in control and IBS subtypes. Values are expressed as mean intensities fluorescence ± SEM. (*) Represents p<0.05 and (**) represents p<0.005 using Mann-Whitney test. Kruskal-Wallis p values are *p = 0.01 (B); p = 0.5 (C); p = 0.95 (D) and *p = 0.03 (E) respectively.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

Techniques: Flow Cytometry, Staining, Control, Fluorescence, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

doi: 10.1371/journal.pone.0042777

Figure Lengend Snippet: Correlation analysis of TLR2 and TLR4 and duration of symptoms according to the subtype of IBS.

Article Snippet: Then, the sections were incubated at 4°C overnight in the same solution supplemented with primary antibodies mouse anti-TLR2 and anti-TLR4 (1∶1,000, Imgenex, San Diego,CA).

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: 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