recombinant human tlr4  (R&D Systems)

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: a , Representative SEM (top) and TEM (bottom) images of Hep3B and Huh7 HCC cell lines after co-culture with K. pneumoniae (MOI = 10). After co-culture of HCC cells with K. pneumoniae , cell lysate was spread on BHI agar for bacterial culture. n = 3 independent experiments with similar results. b , Effect of pasteurized K. pneumoniae (MOI = 10) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). c , Effect of pasteurized K. pneumoniae (MOI = 10) on PCNA expression in HCC cells. n = 3 (Ctrl), 6 (pasteurized KP). d , Screening of K. pneumoniae adhesins by biotin pull-down assay. e , Hep3B membrane protein was incubated with GST or GST–PBP1B together with GST magnetic beads for GST pull-down assay. Corresponding bands in GST–PBP1B and Hep3B cell membrane protein groups were subjected to mass spectrometry analysis. In d and e , n = 3 independent experiments with similar results. f , Binding affinity between PBP1B and TLR4 was detected using SPR. K d , dissociation constant. g , Representative structure of PBP1B and TLR4 after molecular docking (left) and the residues involved in the interaction between PBP1B with TLR4 (right). The structure of TLR4 is coloured pink while the PBP1B is coloured yellow. The light green dash represents hydrogen bond. h , TLR4 from Hep3B and Huh7 cell lysates was pulled down by GST–PBP1B according to the GST pull-down assay. i , Immunoprecipitation of TLR4 from Hep3B or Huh7 cells lysates validated the binding between TLR4 and GST–PBP1B. In h and i , n = 3 independent experiments with similar results. j , Effect of PBP1B (0.05 μM) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). n = 3 independent experiments with similar results. k , Effect of PBP1B (0.05 μM) on TLR4, MyD88, p-P65, P65 and PCNA protein expression in HCC cells, as determined by western blot. n = 3 biologically independent samples. l , m , TLR4i (30 μM) abolished the effect of PBP1B on HCC cell proliferation ( l ) and colony formation ( m ). n = 3 biologically independent samples. n , Effect of K. pneumoniae on TLR4 expression in liver tissues of both germ-free and SPF mice with or without DEN treatment by IHC staining. In b , c , j – m , data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Cell proliferation was analysed using two-way ANOVA. In b , c , j , k , m , comparisons between two groups were analysed using Student’s t -test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Co-Culture Assay, Derivative Assay, Expressing, Pull Down Assay, Membrane, Incubation, Magnetic Beads, Mass Spectrometry, Binding Assay, Immunoprecipitation, Western Blot, Immunohistochemistry

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of K. oxytoca on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KO group n=6; GFD/KP group n=7; GFD/KOP group n=8) and relevant results (b–e) . (b) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (c) Alcian blue staining, E-Cad, CLDN 3, CLDN 1 IHC, and Cy3-conjugated EUB338 probe detection with quantitative analysis. n=6 biologically independent samples. (d) Gut permeability assay using FITC-dextran. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (e) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (f) Design of TLR4i on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KP group n=7; GFD/KPi group n=7) and relevant results (g-k) . (g) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (h) E-Cad IHC and Cy3-conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (i) Gut permeability assay using FITC-dextran. n=6 biologically independent samples. (j) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (k) Cy3-conjugated conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) FISH, TLR4, PCNA, Ki-67 IHC, and Sirius red staining of liver sections. n=6 biologically independent samples. Data (excluding tumor incidence and liver with live bacteria) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Permeability, Bacteria

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of TLR4i on HCC-FMT induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=8; GFD/HCC-FMT group n=15; GFD/HCC-FMTi group n=10) and relevant results (b–g) . TLR4i was daily gavaged in this experiment. (b) Representative images of liver gross morphology (yellow circles indicate tumor area), and H&E staining of mice liver sections with tumor incidence, tumor number, and tumor burden. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). (c) Representative bacterial culture of liver tissues under anaerobic and aerobic conditions with quantitative analysis. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). From left to right and top to bottom, the culture plates are blood agar plate, chocolate blood agar plate, MacConkey agar plate, and Columbia blood agar plate, respectively. Gut permeability assay using 500 kDa FITC-dextran. n=6 biologically independent samples. (d) Representative pictures of Alcian blue staining, E-Cad IHC staining, and Cy3-conjugated EUB338 probe FISH detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (e) Cy3-conjugated EUB338 probe FISH, Cy3-conjugated K. pneumoniae probe FISH, TLR4, PCNA, Ki-67 IHC of liver sections. n=6 biologically independent samples. (f) α-SMA, Desmin IHC staining, and Sirius red staining of liver sections. n=6 biologically independent samples. (g) Mouse Cancer Pathway Finder Array and Inflammatory Response and Autoimmunity PCR Array of liver tissues. n=3 independent experiments with similar results. Data (excluding tumor incidence, liver with live bacteria and PCR array results) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Chocolate, Permeability, Immunohistochemistry, Bacteria

Journal: Frontiers in Pharmacology

Article Title: Melatonin modulates TLR4/MyD88/NF-κB signaling pathway to ameliorate cognitive impairment in sleep-deprived rats

doi: 10.3389/fphar.2024.1430599

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: The primary antibodys used in this experiment are as follows: iNOS(1:2000, BS-0162R, bosterbio), IL-1β(1:2000, BS-0812R, biosscn), TNF-α(1:2000, BA0131, bosterbio), IL-6 (1:2000, BA4339, bosterbio), COX2 (1:5000, ab179800, abcam), IBA1(1:1000, 26177-1-ap, ptgcn), Aβ42 (1:2000, A17911, Abclonal), Occludin (1:5000, 21773-1-Ap, ptgcn), ZO-1 (1:5000, 21773-1-Ap, ptgcn), TLR4 (1:3000, 66350-1-IG, ptgcn), NF-κB p65 (1:2000, bs-20159r, biosscn), IKB-α(1:5000, 10268-1-AP, ptgcn), MyD88 (1:2000, PB9148, bosterbio), p-NF-κB p65 (1:2000, AF 2006, Affinity), p-IKB-α(1:1000, bsm-52169R, biosscn).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Melatonin modulates TLR4/MyD88/NF-κB signaling pathway to ameliorate cognitive impairment in sleep-deprived rats

doi: 10.3389/fphar.2024.1430599

Figure Lengend Snippet: MT regulated brain barrier function and TLR4/MyD88/NF-κB signaling pathway in sleep-deprived rats. (A) Western blot bands showing the protein expression levels of Iba1 and Aβ42 in the HP, respectively. (B, C) Relative protein expression level of Iba1 and Aβ42 in the HP, respectively. (D) Western blot brands showing the protein expression levels of ZO-1 and occludin in the HP, respectively. (E) Western blot brands showing the protein expression levels of TLR4, MyD88, IKB-α, p-IKB-α, NF-κB p65, and p-NF-κB p65 in the HP, respectively. (F, G) Relative protein expression level of ZO-1 and occludin in the HP, respectively. (H–K) Relative protein expression level of TLR4, MyD88, p-IKB-α/IKB-α, and p-NF-κB p65/NF-κB p65, respectively. (L–N) Relative mRNA expression of ZO-1, occluding, and TLR4 in the HP, respectively. The data are expressed as the means ± SEM. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model group.

Article Snippet: The primary antibodys used in this experiment are as follows: iNOS(1:2000, BS-0162R, bosterbio), IL-1β(1:2000, BS-0812R, biosscn), TNF-α(1:2000, BA0131, bosterbio), IL-6 (1:2000, BA4339, bosterbio), COX2 (1:5000, ab179800, abcam), IBA1(1:1000, 26177-1-ap, ptgcn), Aβ42 (1:2000, A17911, Abclonal), Occludin (1:5000, 21773-1-Ap, ptgcn), ZO-1 (1:5000, 21773-1-Ap, ptgcn), TLR4 (1:3000, 66350-1-IG, ptgcn), NF-κB p65 (1:2000, bs-20159r, biosscn), IKB-α(1:5000, 10268-1-AP, ptgcn), MyD88 (1:2000, PB9148, bosterbio), p-NF-κB p65 (1:2000, AF 2006, Affinity), p-IKB-α(1:1000, bsm-52169R, biosscn).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Melatonin modulates TLR4/MyD88/NF-κB signaling pathway to ameliorate cognitive impairment in sleep-deprived rats

doi: 10.3389/fphar.2024.1430599

Figure Lengend Snippet: Mechanistic diagram. Sleep deprivation activates the hippocampal TLR4/MyD88/NF-κB signaling pathway in rats, resulting in hippocampal inflammatory damage and cognitive dysfunction. MT may exert neuroprotective effects by down-regulating the levels of LPS and Aβ42, up-regulating the expression of ZO-1 and Occludin, inhibiting the activation of microglia and the TLR4/MyD88/NF-κB signaling pathway, and reducing the release of pro-inflammatory indicators (IL-1 β, IL-6, TNF-α, iNOS, and COX2) to exert neuroprotective effects and improve chronic sleep deprivation-induced neuroinflammation and cognitive dysfunction.

Article Snippet: The primary antibodys used in this experiment are as follows: iNOS(1:2000, BS-0162R, bosterbio), IL-1β(1:2000, BS-0812R, biosscn), TNF-α(1:2000, BA0131, bosterbio), IL-6 (1:2000, BA4339, bosterbio), COX2 (1:5000, ab179800, abcam), IBA1(1:1000, 26177-1-ap, ptgcn), Aβ42 (1:2000, A17911, Abclonal), Occludin (1:5000, 21773-1-Ap, ptgcn), ZO-1 (1:5000, 21773-1-Ap, ptgcn), TLR4 (1:3000, 66350-1-IG, ptgcn), NF-κB p65 (1:2000, bs-20159r, biosscn), IKB-α(1:5000, 10268-1-AP, ptgcn), MyD88 (1:2000, PB9148, bosterbio), p-NF-κB p65 (1:2000, AF 2006, Affinity), p-IKB-α(1:1000, bsm-52169R, biosscn).

Techniques: Expressing, Activation Assay