Journal: Oncotarget

Article Title: NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells

doi: 10.18632/oncotarget.12355

Figure Lengend Snippet: A. MyD88 mRNA levels in human EOC cell lines (A2780, 01-28, R182, or SKOV3) were measured using reverse transcription real-time PCR. Different letters represent significant difference between two groups ( p < 0.05). B. A2780, 01-28, R182 or SKOV3 cells were treated with vehicle or 30 μM sulindac sulfide (S.S) for 48 h. Cellular lysates were subjected to Western blotting analysis. (C-E) A2780 and R182 cells were treated with vehicle or 10 ng/ml rNAG-1 for 24 h. Cellular mRNA levels were measured using reverse transcription real-time PCR.

Article Snippet: A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells

doi: 10.18632/oncotarget.12355

Figure Lengend Snippet: A. Control or NAG-1 shRNA vector-transfected SKOV3 cells were subjected to Western blot analysis. B. and C. mRNA levels of control or NAG-1 shRNA vector-transfected SKOV3 cells was measured using reverse transcription real-time PCR. A significant difference from the control (*, p < 0.05; **, p < 0.01; or ***, p < 0.001).

Article Snippet: A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, shRNA, Plasmid Preparation, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells

doi: 10.18632/oncotarget.12355

Figure Lengend Snippet: R182 A, C. and SKOV3 B, D. cells expressing the control vector or NAG-1 shRNA plasmid were compared. Cellular lysates were subjected to western blot analysis.

Article Snippet: A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Plasmid Preparation, shRNA, Western Blot

Journal: Oncotarget

Article Title: NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells

doi: 10.18632/oncotarget.12355

Figure Lengend Snippet: R182 A, C. and SKOV3 B, D. cells expressing the control vector or EGFR shRNA plasmid were compared. Cellular mRNA levels were analyzed by reverse transcription real-time PCR. A significant difference from the control (*, p < 0.05; **, p < 0.01; or ***, p < 0.001).

Article Snippet: A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Plasmid Preparation, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells

doi: 10.18632/oncotarget.12355

Figure Lengend Snippet: A. A2780 and R182 cells were treated with 1 μM paclitaxel for 4 days and the spheroids were counted. *A significant difference from the paclitaxel-treated A2780 cells (p < 0.05). B. The viability of EOC cells (A2780, 01-28, R182, or SKOV3 cells) expressing vector or NAG-1 shRNA plasmid were compared. Cells were treated with each dose of paclitaxel for 48 h, and cell viability was measured using MTT assay. *A significant difference from the each vehicle-treated group ( p < 0.05). C. Colonies of EOC cells (A2780 or R182 cells) expressing vector or NAG-1 shRNA plasmid were treated with 1 μM paclitaxel for 48 h and then incubated for 4 days. The number of colonies were counted. *A significant difference from the paclitaxel-treated R182 cells (p < 0.05). D. R182 cells expressing the control vector, SMAD4 shRNA plasmid, or SR-IκB expression plasmid were compared. Each cells were treated with vehicle or 2 μM paclitaxel for 48 h, and cell viability was measured using MTT assay. *A significant difference from only the paclitaxel-treated group (p < 0.05). E. R182 cells were pre-exposed to control, 2 μM 5Z-7-oxozeaenol (TAK-1 inhibitor) or 1 μM AR1478 (EGFR inhibitor) and treated with vehicle or 2 μM paclitaxel for 48 h. *A significant difference from only the paclitaxel-treated group ( p < 0.05).

Article Snippet: A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Plasmid Preparation, shRNA, MTT Assay, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: Decrease in TLR2 signaling after TLR/NLRP3 activation or S.aureus infection is reversed by caspase/NLRP3 inhibition. PMA-primed THP-1 macrophages were stimulated with Pam3CSK4 (0.5 µg/ml) for 4 h and NLPR3 agonists for 2 h. Inhibitors were added 2 h before the inflammasome agonist (A–C) . S. aureus was added 2 h after inhibitors, at the same time as NLRP3 activators and supernatants were sampled 6 h later (D) . Supernatants were collected and levels of cytokines determined using ELISA( ( 1 + x ) n = 1 + n x 1 ! + n ( n − 1 ) x 2 2 ! + ⋯ ). PMA-primed THP-1 macrophages were stimulated with LPS (10 ng/ml) for 4 h and NLRP3 agonists for 2 h. Cell lysates were used for MyD88 detection (E) , while cell supernatants were used for active caspase-1 detection. PMA-primed THP-1 macrophages were stimulated with LPS (10 ng/ml) for 4 h and NLRP3 agonists for 2 h and then stimulated again with 10 ng/ml of LPS for 1.5 h (F-H). Supernatants were collected and levels of cytokines determined using ELISA. Data are represented as mean ± SD of at least 3 replicates (A–D, F–H) . Experiments were repeated at least three times with similar results. One-tailed unpaired t-test was used for statistical analysis, ** p < 0.01, *** p < 0.005.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Activation Assay, Infection, Inhibition, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: Effect of NLRP3 inflammasome activation on MyD88-dependent cytokines and MyD88 cleavage in mouse cells. Mouse BMDMs were first primed with LPS for 3 h; following this, inflammasome activators (1 mM ATP, 2 μM nigericin, 0.5 mg/ml Alum) were added. After 3 h, supernatants were collected and cytokine concentration was determined using ELISA (A, B) . Peritoneal macrophages were primed with LPS for 6 h, after which activators (0.1 mg/ml SiO 2 , 0.5 mg/ml Alum; 20 μg/ml CTB + 2 μg/ml ultrapure LPS) were added. After 1.5 h, 5 h, and 18 h, cells were lysed and blotted for MyD88 (C) . Immortalized wt and GSDMD-KO BMDMs were first primed with LPS for 4 h and stimulated with nigericin for 3 h (D) . Data are represented as mean ± SD of at least 3 replicates (A, B, D) . Representative results of three independent experiments are shown. One-tailed unpaired t-test was used for statistical analysis. * p < 0.05, *** p < 0.005.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: Different caspase-1 cleavage sites in human and mouse MyD88 determine the strength of MyD88-mediated signaling inhibition. HEK293T cells were transfected with human or mouse MyD88 and/or point mutants (0.3 μg or 1 μg) along with increasing amounts of caspase-1 (75/150 ng or 150/300 ng) plasmid. After 24 hours cells were lysed and lysates were blotted for MyD88, while cell supernatants were used for detection of active caspase-1 (A, C, E) . Alignment of human and mouse MyD88 with the indicated position of caspase-1 cleavage site (B) . MyD88-KO HEK293 cells were transfected with human or mouse MyD88 and/or point mutants (0.05 or 0.1 ng/well) along with increasing amounts of caspase-1 (1/2.5 or 10/25 ng/well) and reporter plasmids. Cells were stimulated with 10 ng/ml of interleukin-1β (IL-1β) for 16 h, lysed and NF-κB activation was measured using luciferase assay. Data are represented as mean ± SD of at least 3 replicates (D, F) . Experiments were repeated at least three times with similar results.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Inhibition, Transfection, Plasmid Preparation, Activation Assay, Luciferase

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: LPS/MSU challenge of animals results in decreased cytokine levels in comparison to LPS stimulation. Activation of the inflammasome was triggered in wild-type (wt) (A, B) or Trif -/- knockout (KO) (C, D) mice by the intraperitoneal injection of MSU at 1 mg/animal. Optionally, the NLRP3 inhibitor MCC950 was given intraperitoneally 1 h prior to MSU. Two hours after MSU injection, animals were administered 0.75 mg/kg LPS intraperitoneally. Four hours later, blood was collected for cytokine detection using ELISA (A, C), while peritoneal lavage obtained cells were used for MyD88 detection (B, D). Unpaired two-tailed t-test with Welch’s correction was used for comparison of TRIF-KO populations treated with LPS and MSU/LPS and unpaired two-tailed t-test was used otherwise. ** p < 0.01, *** p < 0.005.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Comparison, Activation Assay, Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: Constitutively present MyD88 cleavage fragment in the Waldenström’s macroglobulinemia (WM) cell line and lack of signaling inhibition by TIR L265P . MWCL-1 cells or B-lymphocytes were lysed and blotted for MyD88 (A) or caspase-1 (D) detection. MWCL-1 cells were seeded into fresh media, and inhibitors were added to the cells for 24 h. Supernatants were collected and the amounts of cytokines determined using ELISA. Data are represented as mean ± SD of at least 3 replicates (B, C) . HEK293 cells were transfected with MyD88 cleavage fragments (hMyD88Δ1-148 or hMyD88Δ1-148 L265P ; 1, 5, 10, 25 ng/well) and reporter plasmids. Cells were lysed after 16 h and NF-κB activation was measured using luciferase assay. Data are represented as mean ± SD of at least 3 replicates (E) . Experiments were repeated at least three times with similar results. One-tailed unpaired t-test was used for statistical analysis, *** p < 0.005.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Transfection, Activation Assay, Luciferase, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

doi: 10.3389/fimmu.2021.790258

Figure Lengend Snippet: A schematic representation of negative feedback regulation of MyD88-mediated signaling through activation of the inflammasome.

Article Snippet: Immunodetection of human MyD88 was carried out using primary anti-MYD88 (ab2) antibody produced in rabbit (Sigma) or MyD88 (D80F5) rabbit mAb (Cell Signaling Technology), phosphorylation of ERK kinases with phospho-p44/42 MAPK (Thr202/Tyr204) E10 mouse monoclonal Ab (Cell Signaling Technology), caspase-1 by anti-caspase-1 (p20 Bally-1) antibody (Adipogen), and tubulin by α/β-tubulin rabbit polyclonal Ab (Cell Signaling Technology) or actin by β-actin (8H10D10) mouse mAb (Cell Signaling Technology) with appropriate secondary antibodies, namely secondary goat polyclonal to rabbit IgG (HRP; Abcam) or goat anti-mouse IgG-HRP antibody (Santa Cruz).

Techniques: Activation Assay

Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: DTHD1 deficiency increased the cytotoxicity of CD161 − CD8 + T EMRA cells . (a) Heatmap showing the correlation of co-expression modules with different cell subsets. (b) Functional annotations of DTHD1-related genes. (c) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0124. (d) Flow cytometric analysis of mean fluorescence intensity (MFI) of Granzyme B of CD161 − CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs after coculture with P815 cell. P value is calculated by Unpaired t-test, P = 0.0012. (e) Structural prediction of DTHD1-MYD88 interaction by ZDOCK. Grey, DTHD1; blue, MYD88; red, mutated regions in DTHD1 . (f) Western blotting analysis of HA in HEK293T cells co-transfected with myc-MYD88 and HA-empty vector or HA-DTHD1 (WT) or HA-DTHD1 (Mutant) expressing plasmids after immunoprecipitated with anti-Myc beads. (g) Boxplot indicating the average expression of MYD88 in CD161 − CD8 + T EMRA cells in HCs and SLE samples from our dataset. P value is from Wilcoxon rank-sum test. (h) Luciferase activity analysis of lysates of HEK293T cells co-transfected luciferase reporter plasmid for NF-κB, pRL-TK-renilla-luciferase plasmid, MYD88 plasmid and WT-DTHD1 plasmid or mutant DTHD1 plasmid ( n = 6). P values are determined by Unpaired t-test. (i) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells pre-treated with DMSO or TAK-242 for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0024. One representative experiment of three is shown (f). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Expressing, Functional Assay, Flow Cytometry, Fluorescence, Structural Proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Luciferase, Activity Assay

Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: CD161 − CD8 + T EMRA cells contribute to SLE by LIGHT signaling . (a) The number (top) and strength (bottom) of interaction among all cells in HCs and SLE samples. (b) Heatmap of differential interactions between HCs and SLE samples in cell–cell communication network. The top bar indicates the sum of incoming signaling and right bar indicates the sum of outgoing signaling. Red indicates increased signaling and blue indicates decreased signaling in SLE. (c) Number of significant ligand-receptor pairs between CD161 − CD8 + T EMRA cells (outgoing) and other cell subclusters (incoming) in HCs (left) and SLEs (right). The relative number of ligand-receptor pairs is represented by the edge width. (d) Comparison of the significant outgoing signaling from CD161 − CD8 + T EMRA cells between HC and SLE. Empty space means the communication probability is zero. P -values are computed from one-sided permutation test. ∗∗∗ P < 0.001. (e) qPCR analysis of TNFSF14 expression in primary CD8 + T cells electroporated with empty vector, DTHD1-WT or DTHD-mut plasmid cocultured with THP1 cells ( n = 3). P values are determined by unpaired t-test. (f) Receiver operating curve for out-of-sample prediction of case–control state by a logistic regression model trained on DEGs in CD161 − CD8 + T EMRA cells. DEGs include IFI27 , IFI44L , RSAD2 , IFI44 , FAM118A , LGALS9 , MX1 , EPSTI1 , USP18 , OAS3 , LAIR2 , IFIT1 , XAF1 . Inset depicts the changes of DEGs in the public transcriptome profile and CD161 − CD8 + T EMRA cells. (g) Graphic abstract showing the expansion of CD161 − CD8 + T EMRA in patients with SLE and DTHD1 downregulation promotes MYD88-mediated expansion and cytotoxicity of this pathogenic CD161 − CD8 + T EMRA subset in SLE.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Comparison, Expressing, Plasmid Preparation, Control

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:

Journal: Cell communication and signaling : CCS

Article Title: MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis.

doi: 10.1186/s12964-024-01930-1

Figure Lengend Snippet: Fig. 1 MyD88D162E mutant mice are protected from DSS-induced acute colitis. Schematic presentation of the DSS-induced colitis experiment. To induce acute colitis, mice were drinking tap water, supplemented with 2% DSS for 8 consecutive days ad libitum. A Disease activity index was defined by observing the stool consistency and the presence of the gross or occult blood. The weight loss was calculated from the weight difference on day1 and day 8 and the colon length was measured from the body of the caecum to the distal part of the rectum. B The part of the colon was washed and IL-6 cytokine was measured using ELISA assay. C Blood was collected and IL-6 was detected in serum using ELISA. D-F Il6, Il1b, and MyD88 mRNA expression was detected using qPCR in colon tissue homogenate. G MyD88 protein detection from obtained colon tissue of treated mice, using Western blot. β-actin was used as loading control. H Schematic presentation of the experiment. LPS was i.p. injected. After 4 h IL-6 and IL-10 cytokines were analyzed in the serum. Combined data from one (B, C. H) or two (A, D-F) indep. exp. are shown as mean ± SEM. LN transformation was applied for the data used in statistical tests (D-F). Studentʾs unpaired t-test was used. p values of < 0.05 (*), < 0.01 (**) are indicated

Article Snippet: To determine the role of caspase-1 in MyD88 signaling regulation cells were transfected with pcDNA3.1 mouse MyD88wt (a gift from R. Medzhitov (Addgene plasmid #13,092; http:// n2t. net/ addge ne: 13092; RRID:Addgene_13092)) or pcDNA3.1 mouse MyD88D162E plasmids (both 0.05 ng/well) w/o increasing concentrations of pCMV mouse Caspase-1 (0.05, 0.1 ng/well) as well as pELAM1-luciferase (NF-κB promotor; 30 ng/well)) (a gift from C. Kirschning, Germany) and phRL-TK Renilla luciferase (3 ng/well) (Promega) for normalization using lipofectamine 2000 transfection reagent (Invitrogen).

Techniques: Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Injection, Transformation Assay

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: Huh7 hepatocytes were transfected with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM). After 48 h, MyD88, IRAK1, IRAK4, and TRAF6 mRNA levels were determined by qPCR ( A , B , C , and D ) and RT-PCR ( E and F ), respectively. Data are presented as the means SD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: ( A ) Sequence alignment of miR-21 and its binding sites in the 3′ UTRs of MyD88 and IRAK1, as predicted by RNA22 software. ( B ) Huh7 hepatocytes (1×10 4 ) were co-transfected with pGL3-Basic, pGL3-MyD88 3′ UTR, or pGL3-IRAK1 3′ UTR firefly luciferase reporter plasmids (80 ng) and pRL-TK Renilla luciferase plasmid (40 ng), together with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM), as indicated. After 48 h, firefly luciferase activity was determined and normalized to Renilla luciferase activity. ( C ) HEK293 cells (1×10 4 ) were co-transfected with GFP control, GFP-MyD88 3′ UTR, or GFP-IRAK1 3′ UTR plasmid (400 ng), together with miR-21 mimics or control RNA (final concentration, 50 nM), as indicated. After 48 h, GFP expression was analyzed by FACS, and the mean fluorescence intensity (MFI) of GFP was determined. ( D and E ) Huh7 hepatocytes (1×10 6 ) were transfected with miR-21 mimics ( D ) or miR-21 inhibitor ( E ) at various concentrations for 48 h ( left ), or at 50 nM (final concentration) for the indicated time ( right ). MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin ( top panel ); MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH ( bottom panel ). Data are presented as the means SD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Sequencing, Binding Assay, Software, Transfection, Luciferase, Plasmid Preparation, Control, Concentration Assay, Activity Assay, Expressing, Fluorescence, Western Blot

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: ( A ) Huh7 hepatocytes were transfected with nonspecific control siRNA or siRNA against MyD88 or IRAK1, as indicated. After 24 h, MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH ( lower panel ); after 48 h, MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin ( upper panel ). ( B ) Huh7 hepatocytes were co-transfected with miR-21 mimic or control RNA and siRNA against MyD88 or IRAK1, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated time, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. ( C ) Huh7 hepatocytes were co-transfected with miR-21 inhibitor or control inhibitor and siRNA against MyD88 or IRAK1 or nonspecific control siRNA, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated times, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. Data are the means SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: During HCV infection, the virus is first recognized by TLRs and RIG-1, which in turn activates MyD88 and IRAK1 to initiate IFN-α synthesis, resulting in the activation of ISGs and the inhibition of HCV replication. In addition, miR-21 expression is activated during HCV infection through two signaling pathways: the PKCε/JNK/c-Jun pathway and the PKCα/ERK/c-Fos pathway. The HCV NS5A protein activates PKCε to enhance the expression of JNK and c-Jun, while the HCV NS3/4A complex stimulates PKCα to promote the production of ERK and c-Fos. The two subunits (c-Jun and c-Fos) of AP-1 join together to recognize the miR-21 promoter and activate the expression of miR-21, which represses the expression of MyD88 and IRAK1 via imperfect base pairing between miR-21 and the 3′UTR of MyD88 and IRAK1. The reduction in MyD88 and IRAK1 causes a reduction of type-I IFN production and ISG expression that might contribute to viral pathogenesis and virus propagation.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Virus, Activation Assay, Inhibition, Expressing, Protein-Protein interactions

Journal: Communications Biology

Article Title: Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells

doi: 10.1038/s42003-019-0432-4

Figure Lengend Snippet: Wnt5a induces the production of pro-inflammatory cytokines in mouse myeloid cells. a Murine RAW264.7 macrophages, or primary (1°) WT C57Bl/6, MyD88 -/- , or Tlr4 -/- BMM, were treated with rWnt5a (6 h). Cytokine levels were determined by CBA. ( n = 4 and n = 3). Dunnett’s test was used for multiple comparisons. b Wnt5a signalling in WT C57Bl/6, MyD88 −/− , and Tlr4 −/− mice. Relative protein levels of MCP-1 (CCL2) or TNFα from the experiment shown ( a ) in different mouse strains are shown. ( n = 3). Dunnett’s test was used for multiple comparisons. c Wnt5a induces Tnf , but not Ccl2 (MCP-1) mRNA transcription as shown by using the transcriptional blocker actinomycin D (ActD) and RAW264.7 cells. The protein levels were determined using CBA. ( n = 3) Dunnett’s test (black annotation) and Holm-Sidak´s test (grey annotations) was used for multiple comparisons. d HA-Wnt5a-induced activation of the mouse Il10 promoter in human THP-1 cells. LPS (18 h) as positive control. ( n = 6). Dunnett’s test was used for multiple comparisons. e Wnt5a-induced IL-10:TNFα protein level ratio in mouse and human cells. ( n = 3 and n = 5). Data were analysed by paired t -test. f Wnt5a-induced activation of an AP-1 reporter in mouse RAW264.7 but inconsistently in human THP-1 cells. ( n = 8) Data were analysed by paired t -test. g Wnt5a signalling in human myeloid cells is inhibited by ectopic expression of dominant negative (DN)-hTLR2 and DN-hTLR4. DN-TLR2, DN-TLR4 or DN-TLR2/DN-TLR4 plasmids, using THP-1 cells and HA-Wnt5a or Ctrl vector. The protein levels were determined using CBA. The ratio rWnt5a-treated/Ctrl-treated for each pair is shown. ( n = 3) Holm-Sidak´s test was used for multiple comparisons. h Wnt5a slightly affects MyD88-independent IRF and NFκB signalling. Overexpression of pcDNA3-HA-Wnt5a (48 h) in THP1-Dual TM MyD-KO cells. ( n = 4) Data were analysed by paired t -test. i Wnt5a signalling in human primary monocytes is blocked by PMB. Relative levels of IL-10, TNFα, IL-6 (CBA), and IL-8 (ELISA) stimulated with rWnt5a (6 h), with or without PMB treatment, are shown. Error bars indicate SEM ( n = 4). Mann–Whitney U test was used for each bar comparison to Ctrl. Error bars indicate SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001. Data were analysed by ANOVA or t -test as indicated for each panel

Article Snippet: For TLR4 or TLR2 inhibition studies, cells were incubated with pAb-hTLR4 (pab-hstlr4), pAb-hTLR2 (pab-hstlr2), or a pAb-Control (pab-sctr) (Invivogen, Toulouse, France) at a concentration of 0.5 μg/ml for 1 h prior to the stimulation, or a MyD88 inhibitor and control peptide set NBP2-29328 (Novus Biologicals, Littleton, CO, USA) or PMB (Sigma–Aldrich).

Techniques: Activation Assay, Positive Control, Expressing, Dominant Negative Mutation, Plasmid Preparation, Over Expression, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Comparison

Journal: Communications Biology

Article Title: Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells

doi: 10.1038/s42003-019-0432-4

Figure Lengend Snippet: Model for Wnt5a as a tolerance-associated molecular pattern (TAMP). The proposed model for the homeostatic Wnt5a (TAMP)-TLR4 feedback inhibition in human myeloid cells (red arrows) or DAMP signal in mouse (yellow arrow). 1 A danger signal [e.g., endotoxin (PAMP) or endogenous DAMP] binds TLR4 during infection or cell damage. 2 The TLR4-induced signal leads to an increased expression of Wnt5a. 3 Wnt5a is produced and released, and binds TLR2 and TLR4. 4 The Wnt5a/TLR-induced signal promotes TLR-MyD88-NFκB p50-homodimer formation and induction of the immunosuppressive cytokine IL-10 (dominant in human) and TLR-MyD88-MAPK-AP-1 (dominant in mouse) inducing TNFα in mouse. 5 IL-10 promotes the inhibition of TNFα and induction of the anti-inflammatory Mo-MDSC/M2 phenotype in human

Article Snippet: For TLR4 or TLR2 inhibition studies, cells were incubated with pAb-hTLR4 (pab-hstlr4), pAb-hTLR2 (pab-hstlr2), or a pAb-Control (pab-sctr) (Invivogen, Toulouse, France) at a concentration of 0.5 μg/ml for 1 h prior to the stimulation, or a MyD88 inhibitor and control peptide set NBP2-29328 (Novus Biologicals, Littleton, CO, USA) or PMB (Sigma–Aldrich).

Techniques: Inhibition, Infection, Expressing, Produced

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 1 | IL-17RD negatively regulates TLR signalling pathways. (a,b) Assay of NF-kB-regulated luciferase reporter activity in HEK293 cells transfected with Myc-tagged IL-17RD (0–100 ng) and (a) MyD88, Mal, TRIF or TRAM (50 ng) with a NF-kB luciferase reporter plasmid (60 ng) or (b) with TRIF (50 ng) and PFR-luciferase (60 ng) with IRF3-Gal4 (30 ng) or IRF7-Gal4 (25 ng) constructs. TK Renilla was measured to determine transfection efficiency. (c,d) Supernatants were measured for (c) IL-8 or (d) RANTES production by sandwich ELISA. (e,f) ELISA of (e) TNF-a or (f) IL-8 from U373 or THP-1 cells, respectively, previously transduced with lentiviral-encoded control or IL-17RD-specific shRNA and treated with LPS (100 ng ml 1) for 24 h. (g) Supernatants from U373 cells stably transduced with control or IL-17RD-specific shRNA measured for RANTES and IP-10 levels by ELISA in response to poly(I:C) (25 mg ml 1). (h,i) Cell lysates from (h) THP-1 cells treated with LPS (100 ng ml 1) for 0, 5, 15, 30, 60 and 240 min and (i) U373 cells treated with poly(I:C) (25 mg ml 1) for 0, 45, 90, 120, 240 and 360 min, both stably expressing control and IL-17RD-specific shRNA, were subjected to immunoblotting with indicated antibodies. (h,i: inset panels) Cell lysates from THP-1 and U373 cells expressing control (Ctrl) or IL-17RD-specific shRNA (shRNA) were analysed for IL-17RD and b-actin expression with specific antibodies by immunoblotting. Overexpressed IL-17RD (oe) was used as a control from HEK293 and U373 cell lysates, respectively. Data are presented (a–g) as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test, *Po0.05; **Po0.01 or (h,i) are representative of three independent experiments. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Transduction, Control, shRNA, Stable Transfection, Expressing, Western Blot, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 4 | SEFIR domain is critical for inhibitory effects of IL-17RD. (a) HEK293 cells were transfected for 24 h with (a) MyD88-FLAG, Mal-FLAG and IL-17RD-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (b) HEK293 cells were transfected for 24 h with MyD88-GFP and IL-17RD-RFP and visualized for expression and localization using confocal microscopy. Cells were also incubated with nuclei-staining DAPI. Confocal images were captured using the 63 objective (oil immersion) on the UV Zeiss 510 Meta System laser scanning microscope equipped with the appropriate filter sets and analysed using the LSM 5 browser imaging software (scale bar, 20 mm). (c) HEK293 cells were transfected for 24 h with MyD88-FLAG with IL-17RD-Myc or IL-17RD DC-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (d) Assay of NF-kB-regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) with IL-17RD-Myc or IL-17RD DC-Myc (50 ng). (e–g) HEK293 cells were transfected for 24 h with (e) TLR4-FLAG and IL-17RD-Myc or IL-17RD DC-Myc, (f) MyD88-FLAG and IL-17RD-Myc, IL-17RD DC-Myc and IL-17RD SEFIR-Myc or (g) TLR4-FLAG and IL-17RD SEFIR-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (h) Assay of NF-kB regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) and IL-17RD-Myc or IL-17RD SEFIR-Myc (100 ng). Data are (a–c,e–g) representative of three independent experiments or (d,h) are presented as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test. *Po0.05. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 9. NS, not significant.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Incubation, Staining, Laser-Scanning Microscopy, Imaging, Software, Luciferase, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 5 | IL-17RD disrupts signalling downstream of TIR adaptors. (a) WT and Il17rd / BMDMs were stimulated for 0, 5, 20 and 60 min with LPS (10 ng ml 1) and cell lysates were subject to immunoprecipitation with an anti-MyD88 antibody and subjected to immunoblotting with indicated antibodies. (b,c) WT and Il17rd / BMDMs or BMDCs were treated for (b) 0, 5, 30 and 60 min with LPS (10 ng ml 1) or (c) 0, 30, 90 and 180 min poly(I:C) (5 mg ml 1), respectively. Cell lysates were subject to SDS treatment, immunoprecipitation with an anti-TRAF6 antibody and probed by immunoblotting with indicated antibodies. (d,e,g) HEK293 cells were transfected for 24 h with MyD88-FLAG with (d) TRAF6-HA with IL-17RD-Myc, IL-17RD DC-Myc or IL-17RD SEFIR-Myc (total 3 mg) or (e) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR DBox1 or IL-17RD SEFIR DBox3 or (g) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (f,h) Assay of NF-kB-regulated luciferase expression in HEK 293 cells transfected with MyD88 (50 ng) with (f) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR DBox1 or IL-17RD SEFIR DBox3 (100 ng) or (h) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R (100 ng). Data are (a–e,g) representative of three independent experiments or (f,h) are presented as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test. *Po0.05; **Po0.01. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 10. NS, not significant.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Immunoprecipitation, Western Blot, Transfection, Luciferase, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 6 | LPS induces in situ association of IL-17RD with MyD88. (a) PBMCs from human donor blood were treated with LPS (100 ng ml 1) for 0, 1, 5, 20 and 60 min and cell lysates were subject to immunoprecipitation with an anti-IL-17RD antibody. Cell lysate and immunoprecipitate samples were immunoblotted with indicated antibodies. (b) PBMCs from human donor blood were transfected with scrambled or IL-17RD-specific siRNA and cultured for 48 h prior to treatment in the absence or presence of LPS (100 ng ml 1) for 20 min. Cells were fixed to slides and subjected to Duolink in situ proximity ligation assay using anti-IL-17RD and anti-MyD88 antibodies. IL-17RD/MyD88 interactions are visible as red fluorescence of the Duolink detection reagents with nuclear staining in blue (DAPI). Images were captured using the 20 objective of a fluorescent microscope with scale bars representing 50 mm. The histogram represents the area of red Duolink signal as a percentage of area of blue DAPI signal from five random images from each of three independent experiments. Probe alone indicates areas of signals from cells subjected to ligation assay, but in the absence of anti-IL-17RD and anti-MyD88 antibodies (five random images from each of the two independent experiments). Data are presented as the mean ±s.e.m. and were subjected to a two-tailed paired Student’s t-test. *Po0.05; **Po0.01. The lowest-right panel demonstrates knockdown of IL-17RD expression by IL-17RD-specific siRNA as determined by immunoblotting of cell lysates from cells treated with LPS with or without prior transfection with IL-17RD-specific siRNA or a scrambled sequence version of this siRNA. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 11.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: In Situ, Immunoprecipitation, Transfection, Cell Culture, Proximity Ligation Assay, Staining, Microscopy, Ligation, Two Tailed Test, Knockdown, Expressing, Western Blot, Sequencing

Journal: PLoS Pathogens

Article Title: Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of Listeria monocytogenes Infection

doi: 10.1371/journal.ppat.1006102

Figure Lengend Snippet: (A) Infectivity of Lm in STAT1 -deficient fibroblasts transduced with lentivirus expressing Fluc (empty circles), MYD88 (red) and UNC93B1 (purple) was tested over a range of MOI. Dose-response curves were fitted to a four-parameter sigmoidal model and EC50 values calculated using GraphPad Prism software: EC50 Fluc = 1.949, EC50 MYD88 = 18.90, EC50 UNC93B1 = 11.11. (B) Diagram illustrating Toll-like receptor (TLR) signaling complex (left) and fragment of planar arrangement of the Myddosome complex (PDB 3MOP) and localization of the MYD88 Ser34 between MYD88 M5 and M6 as well as MYD88 M6 and IRAK I4 1 is shown. (C) Western blot analysis of MYD88 expression in STAT1 -deficient fibroblasts transduced with lentivirus expressing wild type and S34Y mutant MYD88. Equal amounts of each lysate (30μg total protein as measured by BCA assay) were loaded per lane. Actin is shown below as a loading control. (D) NF-κB-luciferase activity in untransduced STAT1 -deficient fibroblasts (negative control, white bar), transduced with lentivirus expressing wild type (WT, black bar), or S34Y mutant MYD88 (S34Y, grey bar) and transfected with the reporter plasmid pNF-κB-luciferase. Error bars represent s.d., n = 3. Statistical significance was determined by one-way analysis of variance (ANOVA) (****, P< 0.0001; n.s., not significant). (E) Infectivity of Lm in STAT1 -deficient fibroblasts transduced with lentivirus expressing Fluc (white bar), wild type MYD88 (WT, black bar), or MYD88 S34Y mutant (S34Y, grey bar). Lm infectivity was measured by flow cytometry and statistical significance determined by one-way ANOVA, error bars represent s.d., n = 3 (****, P< 0.0001, n.s., not significant).

Article Snippet: The following antibodies were used in this study: anti- MYD88 (AF2928, R&D Systems), anti-E-cadherin (BD 610181, BD Biosciences), anti-c-Met (CST 4560, Cell Signaling Technology), anti-actin (a-2066, Sigma Aldrich), goat anti-rabbit (31460, Thermo Fisher Scientific), donkey anti-goat (sc-2020, Santa Cruz Biotech), goat anti-mouse (115-035-146, Jackson ImmunoResearch).

Techniques: Infection, Transduction, Expressing, Software, Western Blot, Mutagenesis, BIA-KA, Control, Luciferase, Activity Assay, Negative Control, Transfection, Plasmid Preparation, Flow Cytometry