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: Molecular medicine reports

Article Title: Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

doi: 10.3892/mmr.2019.10454

Figure Lengend Snippet: Figure 6. HaCaT cells were treated with (A) S100A8 or (B) S100A9 (10 µg/ml) for 24 h and TLR4 expression was increased as assessed by western blotting. Data are expressed as the mean ± standard deviation. **P<0.01 vs. the control. S100, calcium binding protein; TLR4, toll‑like receptor 4.

Article Snippet: Tlr4 inhibitor cli-095 (Tlr4i), protein kinase δ (PKcδ) inhibitor (rottlerin), p38 mitogen-activated Correspondence to: dr in Sik Kim, department of Biomedical laboratory Science, School of Medicine, eulji university, 77 Gyeryoung-ro 771 beon-gil, Jung-Gu, daejeon 34824, republic of Korea e-mail: orientree@eulji.ac.kr Key words: atopic dermatitis, S100 calcium binding protein a8, S100 calcium binding protein a9, keratinocyte, skin barrier protein protein kinase (MaPK) inhibitor (SB202190), MeK inhibitor (Pd98059) and nuclear factor-κB (nF-κB) inhibitor (BaY-11-7085) were obtained from calbiochem (Merck KGaa). antibodies against p38 MaPK (cat. no. 9212), phospho-p38 MaPK (cat. no. 9211), phospho-extracellular signal regulated kinase 1/2 (erK1/2; cat. no. 9101), rabbit igG-HrP (cat. no. 7074), and mouse igG-HrP (cat. no. 7076) were acquired from cell Signaling Technology, inc. anti-erK2 (cat. no. sc-154) and anti-Tlr4 (cat. no. sc-10741) antibodies were obtained from Santa cruz Biotechnology, inc. Production of recombinant S100A8 and S100A9 proteins. in our previous report, the cdna of human S100a8 and S100a9 was cloned into peT28 expression vector (Merck KGaa) (10). recombinant S100a8 and S100a9 expression was induced with 1 mM isopropyl β-d-thiogalactoside in E. coli Bl21 (de3; Merck KGaa).

Techniques: Expressing, Western Blot, Standard Deviation, Control, Binding Assay

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

Journal: Biochemical and biophysical research communications

Article Title: Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes

doi: 10.1016/j.bbrc.2016.07.078

Figure Lengend Snippet: HMGB1 inhibits monocyte apoptosis induced by ABT-737 (A,C) or staurosporine (B,D), which is reversed when monocytes are pretreated with a blocking TLR4 antibody, as evaluated by Annexin V (A,B) and TMRE (C,D) stainings. (E) U0126, a specific MEK/ERK inhibitor, inhibits the effect of rHMGB1 on TMRE fluorescence in monocytes. (F) rHMGB1 (100 ng/ml) induces phosphorylation of ERK in monocytes, which does not occur when monocytes are pretreated with a blocking TLR4 antibody. Data are presented as mean ± SD for N≥4 and at least three separate experiments in all studies. * p<0.05, # p<0.05, ** p<0.01 (Student’s t test).

Article Snippet: When indicated, HMGB1 receptors were blocked on monocytes with anti-human RAGE polyclonal antibody (20 μg/ml, goat IgG), anti-human TLR2 monoclonal antibody (2 μg/ml, mouse IgG2b) or anti-human TLR4 polyclonal antibody (10 μg/ml, goat IgG) (R&D Systems, Wiesbaden, Germany).

Techniques: Blocking Assay, Fluorescence, Phospho-proteomics

Journal: Scientific Reports

Article Title: TLR4 antagonist FP7 inhibits LPS-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection

doi: 10.1038/srep40791

Figure Lengend Snippet: DCs were seeded at 1 × 10 6 cells/ml in fresh medium and stimulated for 24 h with increasing amounts of LPS, in the absence or presence of 10 μM FP7 ( a , b ). Control cells (Ctl) received solvent instead of FP7. Glucose consumption and lactate production were monitored using enzymatic detection kits. Means ± SEM from 5 or 6 experiments are shown. ( c ) DCs were incubated with 10 μg/ml TLR4-blocking antibody or isotype control antibody 1 h prior to LPS addition. Glucose consumption was normalized to non-stimulated control DCs and means ± SEM from 3 experiments are shown. *P < 0.05; # P < 0.01 FP7 compared to Ctl cells.

Article Snippet: Neutralizing mouse IgA 2 anti-human TLR4 and non-specific isotype control antibodies were purchased from InvivoGen.

Techniques: Solvent, Incubation, Blocking Assay

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory Effects of IKK Inhibitor XII, Thymulin, and Fat-Soluble Antioxidants in LPS-Treated Mice

doi: 10.1155/2014/724838

Figure Lengend Snippet: The effects of IKK Inhibitor XII and antioxidant-rich diet on phosphorylation of RelA, IKK, and SAPK/JNK and on the expression of TLR4 and heat shock proteins in the splenic lymphocytes from inflammation-bearing mice. The animal's groups that was indicated in were used (1, control; 2, IB mice; 3, IB + Inh5; 4, IB + Inh10; 5, IB + Inh20; 6, IB + diet; 7, IB + diet + Inh20). Western blot analysis of extracts from isolated mice lymphocytes was provided using corresponding antibodies or anti-GAPDH antibody (bottom). Blot pictures show a single representative experiment from four independent experiments. Histograms below protein bands show protein levels calculated as mean relative units correspondingly to internal control and are the results of blots densitometry by program QAPA from four independent experiments. *Significantly different from control, P < 0.05. ∧ Significantly different from LPS-group, P < 0.05.

Article Snippet: After blocking, the membrane was exposed for 2 hr to antibodies against the following mouse proteins: HSP70 antibody (rabbit anti-mouse HSP 72, clone SPA-812, inducible form, StressGen), HSP90 antibody (rabbit anti-Hsp90 α [Hsp86], StressGen), phospho-NF- κ B antibody (phospho-NF- κ B p65 [Ser 536], #3031, Cell Signaling Technology, Danvers, MA), rabbit phospho- IKK α / β antibody II (Ser 176/180 (Cell Signaling Technology, USA), rabbit phospho-SAPK/JNK antibody to synthetic phospho-peptide SAPK/JNK, or rabbit TLR4 antibody (#2246, Cell Signaling Technology, USA).

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

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the myocardium of chronic heart failure ( CHF ) rats. ( A ) TLR 4 mRNA levels in infarct and remote myocardium of sham and CHF rats (n = 6/group). ( B ) Representative Western blot images and ( C ) quantification of TLR 4 proteins in infarct and remote myocardium of sham and CHF rats (n = 4/group). ( D ) Representative immunohistochemistry images of heart sections stained with TLR 4 (green) and CD 45 (red). The yellow box indicates the enlarged area shown on the right (data are means ± SD , * P < 0.05, ** P < 0.01 versus respective sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the surviving cardiomyocytes of chronic heart failure ( CHF ) rats. ( A ) Representative immunofluorescent images of TLR 4 in cardiomyocytes isolated from sham and CHF rats. ( B ) TLR 4 mRNA levels in cardiomyocytes isolated from sham and CHF rats. ( C ) Representative Western blot images and ( D ) quantification of TLR 4 proteins in cardiomyocytes isolated from sham and CHF rats (data are means ± SD , n = 6/group, ** P < 0.01 versus sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Isolation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4)‐sh RNA lentivirus reduced myocardial inflammation and improved heart function after myocardial infarction ( MI ). The rats received intra‐myocardial injection of normal saline ( NS ), control‐sh RNA lentivirus or TLR 4‐sh RNA lentivirus (1 × 10 9 TU /ml, 100 μl/heart) just after left anterior descending coronary artery ( LAD ) ligation or sham operation. All examinations were performed after 4 weeks of MI . ( A ) Expression of green fluorescent protein ( GFP ; green), the marker gene carried by TLR 4‐sh RNA lentivirus, in the myocardium. The nuclei were counter‐stained with Hoechst 33258 (blue). ( B ) Representative Western blot images and quantification of TLR 4 proteins in sham and chronic heart failure ( CHF ) myocardium. ( C ) tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 protein contents in infarct and remote myocardium. ( D ) Representative images of Masson's trichrome staining (upper panel) and quantification (lower panel) of post‐infarct failing hearts, showing that TLR 4‐sh RNA lentivirus reduced cardiac fibrosis. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with Masson's trichrome reagents. ( E ) Infarct size of post‐infarct failing hearts. ( F ) Fractional shortening (%) of the left ventricle (data are means ± SD , n = 4/group, a P < 0.05, A P < 0.01 versus respective sham‐ NS ; B P < 0.01 versus respective CHF ‐ NS ).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Injection, Saline, Control, Ligation, Expressing, Marker, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Enhanced binding activity of toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes to lipopolysaccharide ( LPS ) and heat shock protein 60 ( HSP 60). Isolated cardiomyocytes were cultured in a CO 2 incubator at 37°C for 24 hrs, then the binding assay was performed at 4°C for 30 min. To block TLR 4, cultured cardiomyocytes were incubated with TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) at 37°C for 15 min., and subsequently incubated with FITC ‐ LPS or OG ‐ HSP 60 at 4°C for 30 min. ( A ) Representative fluorescent images of isolated cardiomyocytes after the incubation with FITC ‐ LPS (green) or OG ‐ HSP 60 (green). ( B ) Binding curves of FITC ‐ LPS to cardiomyocytes. ( C ) Binding curves of OG ‐ HSP 60 to cardiomyocytes.

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Binding Assay, Activity Assay, Isolation, Cell Culture, Blocking Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased cytokine production mediated by toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes. Cultured cardiomocytes from sham and CHF rats were treated with lipopolysaccharide ( LPS ; 1 μg/ml) or heat shock protein 60 ( HSP 60; 1 μg/ml) for 6 hrs. TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) was added 15 min before LPS or HSP 60 treatment. ( A ) Tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 mRNA levels (n = 6/group). ( B ) The amount of TNF ‐α and IL ‐6 released into culture supernatant (n = 6/group). ( C ) Representative Western blot images and quantification of p65 in the nuclei of cardiomyocytes from three independent experiments (data are means ± SD , a P < 0.05, A P < 0.01 versus respective sham; b P < 0.05, B P < 0.01 versus sham‐blank; c P < 0.05, C P < 0.01 versus CHF ‐blank; d P < 0.05, D P < 0.01 versus respective LPS ; e P < 0.05, E P < 0.01 versus respective HSP60).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Cell Culture, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Primers for real‐time PCR

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: