Journal: Frontiers in Immunology

Article Title: Identification of a Heme Activation Site on the MD-2/TLR4 Complex

doi: 10.3389/fimmu.2020.01370

Figure Lengend Snippet: NF-κB Reporter Assays with TLR4, MD-2, and CD14. Human embryonic kidney 293 (HEK293) cells were transfected with plasmid expression vectors for Firefly NF-κB and Renilla luciferase reporters, plus CD14, MD-2, and TLR4 (gray bar); MD-2 and TLR4 (no CD14, orange bar); CD14 and TLR4 (no MD-2, blue bar); or CD14 and MD-2 (no TLR4, green bar). Twenty-four hours after transfection, cells were incubated with media (Control), 10 μM heme, 10 ng/ml LPS, or heme + LPS for 6 h in media containing 1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Results are representative of 3 independent experiments run in triplicate. Bars are means + SD. # P < 0.01 control vs. heme, LPS, and heme + LPS. * P < 0.01 TLR4 + MD-2 + CD14 (gray bar) vs. no CD14 (purple bar), no MD-2 (orange bar), and no TLR4 (green bar) for each stimulant, using One Way ANOVA with Holm-Sidac correction.

Article Snippet: In the morning, cells were transiently transfected with 50 ng TLR4 expression vector (pcDNA3.1-hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid #13086) or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector (pNifty-Luc, InvivoGen), 15 ng of Renilla luciferase control vector (pRL-TK, Promega), 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector (pCDNA3.1-hCD14, a gift from Doug Golenbock, Addgene plasmid #13645) per well using Lipofectamine Plus reagent (LifeTechnology).

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Control, Activity Assay

Journal: Frontiers in Immunology

Article Title: Identification of a Heme Activation Site on the MD-2/TLR4 Complex

doi: 10.3389/fimmu.2020.01370

Figure Lengend Snippet: NF-κB reporter assays with WT and mutant MD-2. HEK293 cells were transfected with plasmid expression vectors for WT or mutant MD-2, TLR4, CD14, Firefly luciferase NF-κB reporter, and Renilla luciferase control. Twenty-four hours after transfection, cells were treated with 10 μM heme or 10 ng/ml LPS for 6 h in media containing 0.1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Luciferase activity is expressed as a percent of cells transfected with WT MD-2 stimulated with heme or LPS (100% activity, black bars and horizontal dashed black line). Magenta cluster and green cluster refer to the 2 clusters of potential heme binding amino acids in . Results are representative of 3 independent experiments run in triplicate. Bar values are means ± SD. ** P < 0.01 vs. WT MD-2 using Kruskal-Wallis One Way Analysis of Variance on Ranks.

Article Snippet: In the morning, cells were transiently transfected with 50 ng TLR4 expression vector (pcDNA3.1-hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid #13086) or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector (pNifty-Luc, InvivoGen), 15 ng of Renilla luciferase control vector (pRL-TK, Promega), 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector (pCDNA3.1-hCD14, a gift from Doug Golenbock, Addgene plasmid #13645) per well using Lipofectamine Plus reagent (LifeTechnology).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Luciferase, Control, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Identification and characterization of zebrafish Tlr4 co-receptor Md-2

doi: 10.1101/817528

Figure Lengend Snippet: A) Schematic representation of LPS transfer from Cd14 to the Tlr4/Md-2 complex. LPS (yellow) is brought by Cd14 (purple) and loaded into Md-2 (navy blue). Md-2 is bound by Tlr4 (cyan). Binding of LPS to the Md-2 co-receptor causes dimerization of the Tlr4/Md-2 complex, activating a downstream inflammatory response. B) The interface between human Tlr4 (cyan) and Md-2 (navy blue) is extensive. Both are required to form a productive interaction with LPS (yellow). Structure shown was made from PDB 3FXI .

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse Tlr4 were obtained from Addgene (#13085 and #13086), originally deposited by Ruslan Medzhitov.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Identification and characterization of zebrafish Tlr4 co-receptor Md-2

doi: 10.1101/817528

Figure Lengend Snippet: A) Maximum likelihood phylogeny for 453 Tlr4 and Cd180 protein sequences. SH supports are indicated on the tree. Wedges are clades, with the length indicating the maximum branch length from the ancestor of the clade. The taxonomic distribution and number of genes within each wedge are indicated on the plot. B-G) Hits for human (purple) and zebrafish (orange) gene sets on six representative chromosomes taken from five species. The species and chromosome are indicated at the top of each plot. The x-axis denotes position on the chromosome. Triangles indicate gene start positions. The green arrow indicates the location of a Tlr4 gene. The y-axis is a running average of the BLAST e-value for each gene set along the genome (see methods). The numbers on the plot indicate the number of human and zebrafish hits within the region shown. H,I) Each row shows the chromosome with the most BLAST hits from the human (panel H) or zebrafish (panel I) gene set. Columns indicate specific genes from the set, with names denoted below. A colored square indicates a gene found somewhere on the chromosome. A green square is a tlr4 gene. The species tree is shown on the left; the chromosome number is on the right. J) Schematic representation of a plausible scenario for the history of the tlr4 gene. Times are taken from Hughes et al. and timetree.org .

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse Tlr4 were obtained from Addgene (#13085 and #13086), originally deposited by Ruslan Medzhitov.

Techniques:

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 5. oxLDL induces uPAR association with TLR4. A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Immunoprecipitation, Immunocytochemistry, Staining, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 6. oxLDL-induced cytokine expression is mediated by NF-κB. A. VSMC were lentivirus-infected to express Gaussia luciferase under control of NF-κB responsive promoter as described in the Materials and methods section. 24 h after infection cells were transfected with SiCo or other siRNA as indicated. 24 h after transfection cells were stimulated with 5 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. B. HEK 293 cells were lentiviral infected to express Gaussia luciferase under control of NF-κB responsive promoter. Expression of uPAR was achieved by lentiviral infection. TLR4 and CD36 expression was achieved by cell transfection. Cells were stimulated with 8 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. C. Cells were stimulated with oxLDL in the presence of proteasome inhibitors. G-CSF and GM-CSF expression was assessed by RT-PCR. D. oxLDL- induced G-CSF and GM-CSF expression in the presence of NF-κB inhibitor BAY-11-7085 was assessed by RT-PCR. E. Primary human monocytes and macrophages were treated with conditioned medium of SiCo- and uPARsi-transfected VSMC stimulated with 8 μg/ml oxLDL. MCP-1 release was followed using Human CCL2/MCP-1 Quantikine ELISA Kit.* indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Expressing, Infection, Luciferase, Control, Transfection, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 7. In vivo evidence for uPAR-mediated events. A. Images captured from sub-fibrous cap area of the plaque stained for CD36 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. B. Images captured from sub-fibrous cap area of the plaque stained for TLR4 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. Scale bar 100 μm. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: In Vivo, Staining