Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: TLR intracellular localization and signaling. TLR3, TLR7, TLR8, and TLR9 are located on the membranes of intracellular compartments, such as the endoplasmic reticulum and endosomes. The myeloid differentiation primary response gene 88 (MYD88) is the one of the functional adapter molecules that have been reported to interact with all TLRs except TLR3. MYD88 recruits IL-IRAK1, IRAK2, IRAK4, and tumor necrosis factor–receptor associated factor 6 (TRAF6), leading ultimately to NF-κB activation and proinflammatory cytokine secretion and an inflammatory response. UNC93B1 is a multitransmembrane-domain-containing protein and plays a critical role in trafficking TLR7 and TLR9 from the endoplasmic reticulum to endosomes, where TLR7 and TLR9 transmit signals via MYD88/TRIF-dependent pathways (Kim et al., 2008).

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Functional Assay, Activation Assay

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: SNP- and estrogen-dependent mRNA expression of TCL1A (A), TLR2 (B), TLR7 (C), TLR9 (D), TLR10 (E), MYD88 (F), and UNC93B1 (G) in LCLs (H). Western blot analysis was performed for TCL1A, TLR2, TLR7, TLR9 TLR10, MYD88, UNC93B1, and ACTB in LCLs with known TCL1A SNP genotypes. The cells were treated with 0.1 nM E2 or with 0.1 nM E2 plus 10−7µM 4-hydroxytamoxifen (4OH-TAM) for an additional 24 hours. Eight cell lines homozygous for the variant (V) genotypes for all three of the TCL1A SNPs and eight cell lines homozygous for WT genotypes were used in these experiments. ***P < 0.0001.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing, Western Blot, Variant Assay

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: Correlations of TCL1A mRNA expression and those of toll-like receptors and MYDBB in the Human Variation Panel of 300 LCLs

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: Correlations of MYD88 mRNA expression with those of toll-like receptors and UNC93B1 in the Human Variation Panel of 300 LCLs

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: TCL1A could modulate TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 expression in LCLs. Relative mRNA expression (A and B) of TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of TCL1A in LCLs with known TCL1A SNP genotypes using pooled siRNA. Eight cell lines of each genotype were used in these experiments. *P < 0.05. Student’s t test was performed to compare gene expression in LCLs with differing TCL1A SNP genotypes before and after gene knockdown, *P value ≤ 0.05 was considered statistically significant. All values are mean ±S.E.M for three separate independent assays. Protein expression was determined by Western blot analysis (C). Relative mRNA expression (D and E) of TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of MYD88 in LCLs with known TCL1A SNP genotypes using pooled siRNA. (F) Western blot analysis was performed for TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of MYD88.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing, Knockdown, Western Blot

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: TCL1A SNP- and estrogen-dependent NF-κB activation as determined by NF-κB reporter assays could be altered by the knockdown or inhibition of MYD88. ER blockade by fulvestrant (ICI) or 4-hydroxytamoxifen (4OH) treatment resulted in TCL1A SNP -dependent NF-κB activation (A and B). TCL1A SNP and estrogen-dependent NF-κB activation could be blocked by MYD88 siRNA knockdown (C) or by exposure to MYD88 inhibitory peptide (100 µM) (D). Specifically, LCLs were cotransfected with an NF-κB reporter construct and siRNA (MYD88 or control siRNA); 24 hours after transfection, cells were treated with either vehicle or 0.1 nM E2 for 24 hours, followed by 10−7µM 4OH or ICI for an additional 24 hours. In some experiments, cells were exposed to MYD88 inhibitory peptide for 24 hours before E2 treatment. Luciferase activity was measured 72 hours after transfection. The firefly luciferase activity derived from the NF-κB responsive reporter was normalized by the use of Renilla luciferase activity as a control to correct for possible variation in transfection efficiency. All experiments were repeated three times in triplicate. **P < 0.001. (E) Coimmunoprecipitation was used to determine whether TCL1A protein could interact with MYD88 in LCLs. Whole-cell lysates from 1 × 107 LCLs were immunoprecipitated with anti-TCL1A (1:50) antibodies or anti-IgG antibodies. Whole-cell lysate (input, left panel) and immunoprecipitated samples (middle and right panels) were immunoblotted and probed with antibodies against TCL1A and MYD88.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Activation Assay, Knockdown, Inhibition, Construct, Control, Transfection, Luciferase, Activity Assay, Derivative Assay, Immunoprecipitation

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 3 Soyasaponins inhibited the recruitments of TLR4 and MyD88 into lipid rafts in liver tissues. The lipid rafts (fraction 3 and 4) were fractionated from liver tissue lysates by using sucrose gradient ultracentrifugation. The protein levels of flotillin-1 (a lipid raft marker), TLR4 and MyD88 were detected by western blotting. The ratio of the amount of TLR4 (or MyD88) in lipid raft to that in total fractions were quantitatively calculated. Results reported are Means ± SD of samples from six mice in each group (n = 6). Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. LPS alone

Article Snippet: Antibodies for MyD88, β-actin and all secondary antibodies used for western blotting were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Marker, Western Blot, Software, Control

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 4 Effects of soyasaponins on the protein levels of molecules in TLR4/MyD88 signaling pathway in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pre-treated with graded concentrations (10, 20 or 40 μmol/L) of soyasaponins (A1, A2, or I) for 2 h and then stimulated with LPS (1 μg/mL) for 30 min (a and b), 1 h (c), or 3 h (d and e). The levels of molecules (MD-2, TLR4, TIRAP, MyD88, p-IRAK4, p-IRAK1 and TRAF6) in TLR4/MyD88 signaling pathway were measured by western blotting. Results reported are Means ± SD of three independent experiments. Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. LPS alone

Article Snippet: Antibodies for MyD88, β-actin and all secondary antibodies used for western blotting were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Software, Control

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 5 Soyasaponins inhibit the protein expression of MyD88 and TRAF6, and activation of NF-κB in MyD88-transfected HEK293T cells. HEK293T cells were transfected with MyD88-flag plasmid for 24 h, and then treated with graded concentration (20 or 40 μmol/L) of soyasaponin (A1, A2 or I) or ST2825 (a MyD88 inhibitor) for 6 h. The protein levels of TLR4 (a), MyD88 (b), TRAF6 (c), p-p65 and p65 (d) were measured by western blotting. Results reported are Means ± SD of three independent experiments. Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. MyD88-flag plasmid transfected group

Article Snippet: Antibodies for MyD88, β-actin and all secondary antibodies used for western blotting were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Software, Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 2. Effect of ox-LDL on the TLR4 signaling pathway in MOVAS cells. (a) mRNA expression of TLR4, TIRAP and MyD88 was measured by qRT-PCR. *p < .05 compared with other concentrations of ox-LDL groups or control group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (b) *p < .05 compared with either time point or control groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 3. Effect of corilagin on the TLR4 signaling pathway in MOVAS cells stimulated by ox-LDL. (a) mRNA expression of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO and IRF5 was measured by qRT-PCR. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (B. C) Protein abundance was measured by western blotting. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group, as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (D, E) Abundance of IL-6 and MCP-1 in cell culture supernatant was measured by ELISAs. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (F) Effect of corilagin on the MOVAS cells proliferation. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (G, H) apoptosis ratio of MOVAS were detected by Annexin V staining. No significant difference was found among four groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Cell Culture, Staining

Journal: mBio

Article Title: Plasmodium falciparum Histidine-Rich Protein II Compromises Brain Endothelial Barriers and May Promote Cerebral Malaria Pathogenesis

doi: 10.1128/mBio.00617-16

Figure Lengend Snippet: HRPII activates an inflammatory pathway in human cerebral microvascular endothelial cells. (A) qRT-PCR of chemokine/cytokine mRNA levels of hCMEC/D3 cells treated with 25 µg HRPII or BSA for 8 h and 24 h. (B) TEER measurements for in vitro hCMEC/D3 barriers transfected with shRNAs for NFκB (N1 and N3) or a scrambled control (Scrb) for 36 h or incubated with inhibitors for NFκB, celastrol (Ce), and triptolide (tr) for 2 h prior to addition of HRPII (H; 10 µg). Data are mean values ± SEM of results from 6 to 8 replicates pooled from three independent experiments. ***, P < 0.0001 (by one-way ANOVA). (C) TEER measurements for in vitro barriers transfected with shRNAs to MyD88 (M1 and M3 and M5) or a scrambled control (Scrb) for 36 h prior to addition of recombinant purified HRPII (10 µg). Data are mean ± SEM of results from 6 to 8 replicates pooled from 3 independent experiments. ***, P < 0.0001 (by one-way ANOVA). Results of assessment of knockdown levels are shown in Fig. S2 in the supplemental material. (D) TEER measurements for in vitro barriers transfected with shRNAs for caspase-1 (C1 and C2) or a scrambled control (Scrb) for 36 h or with IL-1Ra (500 ng), anti-IL-1β (αIL-1β) (25 ng), or the caspase-1 inhibitor YVAD-CMK (80 µM) (C1 Inh) for 1 h prior to treatment with recombinant purified HRPII (10 µg; H). Data are mean values ± SEM of results from 6 to 8 replicates pooled from four independent experiments. ***, P < 0.001 (by one-way ANOVA); **, P < 0.05 (by one-way ANOVA). (E) Quantitative ELISA for cleaved IL-1β from cell lysates. Cells were treated for 24 h with HRPII (10 µg), LPS (3 µg/ml), or IFN-γ (100 ng/ml) or left untreated. Data represent results from three biological replicates, each performed in triplicate. *, P = 0.0002; **, P = 0.0005 (compared to untreated control by unpaired t test).

Article Snippet: Cells were then incubated for 36 h. HRPII was then added, and TEER measurements were recorded over 24 h. shRNAs for each gene were purchased from Origene as follows: for the Myd88 gene, TG311320; for the NFκB gene, TR318700; for the caspase-1 gene, TG305640; for the TLR9 gene, TR301076; for the TLR5 gene, TR308792; and for the TLR2 gene, TR320553.

Techniques: Quantitative RT-PCR, In Vitro, Transfection, Incubation, Recombinant, Purification, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Plasmodium falciparum Histidine-Rich Protein II Compromises Brain Endothelial Barriers and May Promote Cerebral Malaria Pathogenesis

doi: 10.1128/mBio.00617-16

Figure Lengend Snippet: Model for HRPII recognition by human brain endothelial cells and the intracellular pathway that leads to BBB leakage. HRPII binds to an as-yet-unidentified receptor and may be internalized . Inflammasome adaptor proteins (likely ASC/CARD) associate with this endosome and recruit procaspase-1 (Pro-casp-1), which is auto-catalytically activated . Active caspase-1 can cleave pro-IL-1 into its mature form . Mature IL-1β is secreted , such that it can then bind to the IL-1 receptor, IL-1R . Signaling through MyD88, IL-1R activates NFκB , as does downstream signaling from the inflammasome . NFκB mediates transcription of inflammatory genes , resulting in a redistribution of tight junction and adherens junction proteins and a compromised blood-brain barrier , as well as in an increase in levels of surface adhesion molecules .

Article Snippet: Cells were then incubated for 36 h. HRPII was then added, and TEER measurements were recorded over 24 h. shRNAs for each gene were purchased from Origene as follows: for the Myd88 gene, TG311320; for the NFκB gene, TR318700; for the caspase-1 gene, TG305640; for the TLR9 gene, TR301076; for the TLR5 gene, TR308792; and for the TLR2 gene, TR320553.

Techniques: