Journal: bioRxiv

Article Title: Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells

doi: 10.1101/2020.04.03.024471

Figure Lengend Snippet: (A) TLR2 knockdown effect on IL-8 production induced by lipopeptides in A549 cells. siRNA-transfected cells were treated with either pam2CSK4 or pam3CSK4 for additional 24 h. (B) Blocking antibodies for TLR2, TLR4, and CD14 had no effect on lipopeptide-induced IL-8 production. Cells were treated with blocking antibodies for 1 h prior to the lipopeptide stimulation for 24 h. Mouse IgG2aκ (for TLR2 and TLR4 antibodies) and IgG1aκ (for CD14 antibody) were used as isotype controls. Bars indicate means ± S.D. (n=3). (C) Intracellular localization of TLR2 in A549 cells was determined by immunostaining and FACS. Grey areas indicate isotype control. Dotted lines indicate fixed cells for surface staining and closed lines indicate permeabilized cells for intracellular staining of TLR.

Article Snippet: Functional grade monoclonal antibodies for blocking TLR2 (TL2.1), CD14 (61D3) and TLR4 (HTA125) and their matching isotype antibodies were purchased from eBioscience (San Diego, CA, USA).

Techniques: Transfection, Blocking Assay, Immunostaining, Staining

Journal: bioRxiv

Article Title: Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells

doi: 10.1101/2020.04.03.024471

Figure Lengend Snippet: (A) A role of cholesterol-enriched lipid-raft in lipopeptide-induced IL-8 secretion in A549 cells. (B) A role of clathrin-mediated endocytosis in lipopeptide-induced IL-8 secretion. A549 cells were pre-treated with either MβCD or chlorpromazine for 1 h in indicated concentration and then stimulated with lipopeptides. (C) A role of macropinocytosis in lipopeptide-induced IL-8 secretion in A549 cells. Cells were pre-treated with various concentrations of cytochalasin D for 1 h and then lipopeptides-containing media were added for stimulation. (D) An effect of soluble CD14 (sCD14) on lipopeptide-induced IL-8 secretion. sCD14 were treated for 1 h prior to lipopeptide treatment (100 ng/ml Pam2CSK4 or 10 μg/ml Pam3CSK4). IL-8 secretion in the supernatant was determined by ELISA. Bars indicate means ± S.D. (n=3). (E) TLR2 signaling activity in CHO/CD14/TLR2 cells upon lipopeptide stimulation. Cells were treated with indicated concentrations of pam2CSK4 or pam3CSK4 for 24 h and then analyzed by flow cytometry for CD25 expression. Mean fluorescence intensity (MFI) is shown.

Article Snippet: Functional grade monoclonal antibodies for blocking TLR2 (TL2.1), CD14 (61D3) and TLR4 (HTA125) and their matching isotype antibodies were purchased from eBioscience (San Diego, CA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Flow Cytometry, Expressing, Fluorescence

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells ( control ). Mean ± SEM of six independent experiments are given

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control, Activity Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced PGE 2 synthesis. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 and EGFR, or the COX inhibitor indomethacin ( indo ) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE 2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells ( control ). Mean ± SEM of six independent experiments are given

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control, Activity Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced PGE 2 synthesis. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 and EGFR, or the COX inhibitor indomethacin ( indo ) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE 2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control

Journal: bioRxiv

Article Title: Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells

doi: 10.1101/2020.04.03.024471

Figure Lengend Snippet: (A) TLR2 knockdown effect on IL-8 production induced by lipopeptides in A549 cells. siRNA-transfected cells were treated with either pam2CSK4 or pam3CSK4 for additional 24 h. (B) Blocking antibodies for TLR2, TLR4, and CD14 had no effect on lipopeptide-induced IL-8 production. Cells were treated with blocking antibodies for 1 h prior to the lipopeptide stimulation for 24 h. Mouse IgG2aκ (for TLR2 and TLR4 antibodies) and IgG1aκ (for CD14 antibody) were used as isotype controls. Bars indicate means ± S.D. (n=3). (C) Intracellular localization of TLR2 in A549 cells was determined by immunostaining and FACS. Grey areas indicate isotype control. Dotted lines indicate fixed cells for surface staining and closed lines indicate permeabilized cells for intracellular staining of TLR.

Article Snippet: Functional grade monoclonal antibodies for blocking TLR2 (TL2.1), CD14 (61D3) and TLR4 (HTA125) and their matching isotype antibodies were purchased from eBioscience (San Diego, CA, USA).

Techniques: Transfection, Blocking Assay, Immunostaining, Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Activation of COX-2 and release of PGE 2 in A549 cells and human lung cancer tissue in response to LPS. a Effect of COX inhibitors on LPS-induced proliferation of A549 cells in vitro. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of indomethacin ( indo ) or the COX-2 inhibitor NS-398. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells ( control ). Mean ± SEM of six independent experiments are given. b Expression of COX-2 mRNA in response to LPS in A549 cells and human lung cancer tissue. A549 cells and specimen of human adenocarcinoma were either sham-incubated or exposed to 10 μg/ml LPS F515. After 16 h, mRNA was extracted and subjected to quantitative reverse transcriptase polymerase chain reaction. The ΔΔCT values represent relative expression of COX-2 mRNA normalized to the internal reference HPRT mRNA in LPS versus unstimulated A549 cells or lung adenocarcinoma ( Adeno ) tissue. Mean values ± SEM, originating from four independent experiments, each performed in duplicate are given. c Release of PGE 2 in A549 cells in response to LPS. A549 cells were either sham-incubated ( control ) or exposed to the given concentrations of LPS (at least n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total at least n = 6) for various time periods. 8 h before the end of the incubation period, and AA was added. PGE 2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Activation Assay, In Vitro, Incubation, Control, Activity Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

doi: 10.3390/ijms222111560

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: For the blockade of TLR2 and TLR4, ATRA-induced differentiated HL-60 cells and neutrophils were pretreated with 10 μg/mL functional graded monoclonal mouse anti-human TLR2 (clone TL2.1) antibody (Cat. # 16-9922-82, eBioscience, San Diego, CA, USA), 10 μg/mL functional graded monoclonal mouse anti-human TLR4 (clone HTA125) antibody (Cat. # 16-9917-82, eBioscience), or 10 μg/mL functional graded mouse IgG 2a , kappa monoclonal (clone eBM2a) isotype antibody (Cat. # 16-4724-85, eBioscience) at 37 °C for 30 min.

Techniques: Concentration Assay