Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin deficiency inhibited TLR4/JNK/NF-κB signaling activation in renal IR injury A TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in kidneys of WT and mindin KO mice induced by IR or not. B – E Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus WT Sham or KO Sham. # P < 0.05 versus WT IR

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin overexpression promoted production of inflammatory mediators and activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A Representative photomicrographs showing the immunofluorescence of GFP in adGFP and adMindin cells. B Mindin protein expression in adGFP and adMindin cells. C – E Protein levels of TNF-α and MCP-1 in the media of cultured TECs by Western blot. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. F TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in adGFP and adMindin cells induced by HR or not. G – J Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. K Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in mindin-overexpressed HK-2 cells. ***P < 0.001 versus adGFP HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Over Expression, Activation Assay, In Vitro, Immunofluorescence, Expressing, Cell Culture, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin knockdown inhibited the activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. B – F Quantitative analysis of TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. * P < 0.05, ** P < 0.01 versus HR. G Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. ***P < 0.001 versus control HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Knockdown, Activation Assay, In Vitro, Western Blot, Expressing, Control

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Co-immunoprecipitation of TLR4 and mindin in HK-2 cells. Lysates of HK-2 cells were incubated with anti-TLR4 antibody and immune complexes were precipitated by protein A/G beads. The TLR4 complex was determined TLR4 and mindin proteins in the complex. The whole cell lysate was used to detect TLR4 and mindin by Western blot

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Immunoprecipitation, Incubation, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Binding assy of mindin to TLR4 proteins by SPR. Different concen-trations of TLR4 protein (3.125, 6.25, 12.5, 25.0, 50.0, 100.0 nM) were performed to analyse the binding and dissociation rate constants between mindin and TLR4 proteins. The binding constant of them was calculated to evaluate protein binding

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Binding Assay, Protein Binding

Journal: International Journal of Molecular Sciences

Article Title: Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation

doi: 10.3390/ijms23021003

Figure Lengend Snippet: TLR2-dependent immune activation in the in vitro MLN model. Horizontal axis indicates the concentrations of inhibitors (μM) (C29: TLR2 inhibitor; TAK242: TLR4 inhibitor); vertical axis represents the levels of IL-10 in the co-culture supernatant. Adjust R square was calculated within the linear range. All tests were replicated three times.

Article Snippet: To investigate the effect of TLR2 and TLR4 in the present in vitro MLN model, inhibitors of TLR2 (C29) and TLR4 (TAK242) were purchased from MedChemExpress [ , ].

Techniques: Activation Assay, In Vitro, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4 engagement and signalling involved in HAdV-lactoferrin DC uptake and maturation. (A) HAdV-lactoferrin complexes were incubated with recombinant TLR4/MD-2 for 30 min. and then added to DCs. Uptake was quantified 24 h post-incubation by flow cytometry (n =11, statistical analyses by two-tailed paired t-test); (B) DCs were pre-treated for 1 h with TAK-242, HAdV-lactoferrin complex uptake was analysed 24 h post-incubation by flow cytometry (n = 6, statistical analyses by two-tailed paired t-test); (C) IL-1β release following pre-treatment with TAK-242 (n ≥ 3 statistical analyses by t-test); (D) TNF levels 24 h post-incubation ± TAK-242 (n ≥ 3, statistical analyses by two-tailed paired t-test); (E) Percent infection following inhibition with Pepinh-TRIF (n ≥ 6, statistical analyses by two-tailed paired t-test); (F) Percent infection following inhibition with oxPAPC (n ≥ 6, statistical analyses by two-tailed paired t-test); (G) IL-1β release from DCs incubated with HAdV-lactoferrin ± oxPAPC and Pepinh-TRIF (n = 3, statistical analyses by two-tailed paired t-test).

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Incubation, Recombinant, Flow Cytometry, Two Tailed Test, Infection, Inhibition

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4-mediated HAdV-lactoferrin uptake in DCs and IL-1β release Lactoferrin binds to HAdV capsid and retargets the capsid toward TLR4 complex on the cells surface. Following TLR4 engagement, its TIR domain recruits MyD88 and TIRAP, which bridge TLRs to IRAK and MAPK family members that activate NF-kB, AP-1, and IRF. This priming event initiates transcription of genes coding for inflammasome components (e.g., NLRP3 and IL-1β). Under prototypic conditions, DCs detect a second perturbation (signal 2) that induced ROS release (mitochondrial stress) or K + efflux (perturbations of cellular integrity), and/or cathepsin B release from lysosome rupture. These pathways do not appear to be activated by HAdV-lactoferrin complexes. In addition to TLR4 pathways, the RIPK1-RIPK3 pathway is activated through an autocrine-TNF release. During inflammasome formation, pro-caspase-1 auto-activation induces cleavage of pro-IL-1β and likely GSDMD, which will initiate, but not complete, the loss of plasma membrane integrity via pore formation, allowing IL-1β release. Twenty-four hours post-challenge, DC membrane integrity is intact, consistent with the involvement of ESCRT-III complex and repairing GSDMD pores.

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Activation Assay, Clinical Proteomics, Membrane