Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Bone marrow derived macrophages (A, B) were stimulated with increasing concentrations of S-LPS in presence of 10 µg/mL of PIM 2 mimetic or deAcPIM 2 mimetic, or vehicle control, and TNF (A) and IL-12 p40 (B) were measured in supernatants after overnight incubation. Results are mean +/− SEM from n = 6 mice from three independent experiments. PIM analogues were titrated (C, D) in the presence of 0.1 µg/mL S-LPS and a 10 µg/mL dose was chosen as this concentration was sufficient for the active PIMs to strongly inhibit LPS-induced TNF (C) and IL-12 p40 (D) release without cytotoxicity. (E–J) HEK cells stably transfected with TLR4, MD2 and CD14 were incubated with PI (E), PIM 1 (F), isoPIM 1 (G), deAcPIM 2 mimetic (H) or PIM 2 mimetic (I) (10 µg/mL; dotted line) prior to incubation with biotinylated S-LPS (2,5 µg/mL) and streptavidin FITC (black line) or only streptavidin FITC (grey histogram). Non transfected HEK cells were incubated with biotinylated S-LPS as a control (J). Results are from one experiment representative of three independent experiments. (K) Percentage of S-LPS-binding to HEK-MTC cells in presence of vehicle, PI, isoPIM 1 , deAcPIM 2 mimetic or PIM 2 mimetic. Results are the mean +/− SD from three independent experiments. (L) Human IL-8 was measured in the supernatant after overnight S-LPS stimulation of HEK-MTC cells. Results are mean +/− SD from triplicates, from one experiment representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle; θ, p<0.05; θθθ, p<0.001 indicate significant differences between PIM 1 or isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic and deAcPIM 2 mimetic as control.

Article Snippet: PIMs were incubated in presence or absence of 5 μg/mL recombinant mouse CD14 Fc chimera (endotoxin <1.0EU/μg protein; R&D system).

Techniques: Derivative Assay, Control, Incubation, Analogues, Concentration Assay, Stable Transfection, Transfection, Binding Assay

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 mice (A, C) or CD14 KO mice (B, D) were incubated with synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL) or control vehicle prior to stimulation with Malp2 (30 ng/mL; A, B) or Pam 3 CSK 4 (Pam 3 ; 0.5 µg/mL; C, D). TNF release was measured in supernatants after overnight incubation. Results are mean +/− SD from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; †, p<0.05; †††, p<0.001, indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: PIMs were incubated in presence or absence of 5 μg/mL recombinant mouse CD14 Fc chimera (endotoxin <1.0EU/μg protein; R&D system).

Techniques: Incubation, Control

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 or CD14 KO mice were stimulated with increasing concentrations of S-LPS (A, B) or Re-LPS (C, D). TNF (A, C) and IL-12 p40 (B, D) concentrations were measured in the supernatants after overnight incubation. Results are mean +/− SEM from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences between C57Bl/6 and CD14 KO.

Article Snippet: PIMs were incubated in presence or absence of 5 μg/mL recombinant mouse CD14 Fc chimera (endotoxin <1.0EU/μg protein; R&D system).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Concentrations of TNF (A–C) and IL-12 p40 (D–F) in supernatants of wild type (A, B, D, E) or CD14-deficient (C, F) macrophages stimulated overnight with 100 ng/mL of S-LPS (A, D) or Re-LPS (B, C, E, F) in the presence of synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL), or vehicle. Results are mean +/− SD from n = 4 mice from two independent experiments representative of three independent experiments. ND: not detected. **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: PIMs were incubated in presence or absence of 5 μg/mL recombinant mouse CD14 Fc chimera (endotoxin <1.0EU/μg protein; R&D system).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: A schematic representation of our experimental approach showing rTLR4 conjugation to magnetic nanoparticles and its in vitro complexation with LPS in the presence of free forms of MD2 and CD14 co-receptors. The bottom left panel shows the actual optical micrographs of MP-rTLR4-MD2-LPS (test) and MP-rTLR4 (control) complexes recorded in brightfield and complementary fluorescence modes at two different emission wavelengths: 525 nm (green) for alexafluor-tagged LPS and 647 nm (red) for alexafluor-tagged MD2.

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Conjugation Assay, In Vitro, Fluorescence

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: (A) LPS monomerization aided by surfactant Tween 20 addition. (B) The importance of CD14 and MD2 on the extent of complexation, and (C) the molar amounts of molecules used in these reactions. (D) Cross-validation of LPS capture using the LAL assay. (E–H) Sequential scouting experiments: Bulk concentrations of LPS (E) , rTLR4 (F) , MD2 (G) , and CD14 (H) were optimized one-by-one and the optimized values were fixed in all the subsequent experiments to get maximum LPS capture efficiency at each step. The fixed concentration values of the remaining reagents (all in nM) are listed on top of each graph. All experiments were performed in 0.5% PBST, pH 7.4 ( n = 3) (SN = supernatant, ns, p >0.05, * p = 0.01–0.05, ** p = 0.001–0.01, *** p < 0.001).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: LAL Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: (A) Time-lapsed measurements of LPS, MD2, and CD14 concentrations showing molecular binding kinetics and the role of CD14 as a catalyst. While the LPS and MD2 molecules were measured using their fluorescence intensity in the SN, CD14 was quantified via silver staining. (B) CD14 visualized after alexafluor tagging (λ em = 647 nm). The complementary brightfield and fluorescence optical images showed CD14 in the bound state only in the middle of the reaction cycle. (C) Varying the sequence of molecular addition had little impact on the extent of LPS capture. Here, sequential addition implies prior incubation of CD14 with LPS and MP-rTLR4 with MD2 followed by their rapid mixing as opposed to co-incubation of all the reagents from the beginning ( n = 3) (SN = supernatant).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Binding Assay, Fluorescence, Silver Staining, Sequencing, Incubation

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: Inhibitory ligand screening. Results shown for LPS-interacting alexidine and sushi molecules used as test, and LPS-non-interacting bacitracin and vancomycin molecules used as control ( n = 3). While the LPS fluorescence intensity in the SN remained constant upon addition of MP-rTLR4-MD2-CD14 assay mix (marked here as “A”) in the presence of alexdine and sushi, it reduced significantly with bacitracin and vancomycin suggesting the inhibitory potential of the first two molecules for obstructing rTLR4 pathway. All the data have been normalized with respect to the fluorescence intensity before “A” addition. The concentrations of each ligand used in reaction was 2.4 μM in their respective reactions (SN = supernatant, ** p = 0.001–0.01, *** p < 0.001).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

doi: 10.1074/jbc.M117.791780

Figure Lengend Snippet: FNC-RED induces NF-κB activation via murine TLR4/MD-2 in Ba/F3 transfectant cells. A, chemical structure of FNC-RED determined by NMR spectroscopy. B, Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were treated with anti-mouse TLR4/MD-2 mAb (clone MTS510) (20 μg/ml) or isotype control antibody (Ct. Ab) (20 μg/ml) for 1 h and subsequently stimulated with the indicated concentration of FNC-RED for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Dashed lines and open histograms depict those cultured with medium alone and FNC-RED, respectively. Filled histograms depict those cultured with FNC-RED in the presence of anti-mouse TLR4/MD-2 mAb or isotype control antibody. Black and gray values depict mean fluorescence intensity (MFI) of GFP expression in cultured cells stimulated with FNC-RED and FNC-RED with indicated Abs, respectively. C, chemical structure of FNC (left). Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were stimulated with FNC (50 μg/ml) for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Open and filled histograms depict those cultured with medium alone and FNC, respectively (right). Black and gray values depict the MFI of GFP expression in cultured cells stimulated with medium alone and FNC, respectively. D, Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were treated with polymyxin B (1 μg/ml) for 1 h and subsequently stimulated with the indicated concentration of lipid A or FNC-RED for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Filled and open histograms depict those cultured with lipid A or FNC-RED in the presence and absence of polymyxin B, respectively. Dashed lines depict those cultured with medium alone. Black and gray values depict MFI of GFP expression in cultured cells stimulated with lipid A or FNC-RED and medium, lipid A, or FNC-RED with polymyxin B, respectively. E, Biacore sensorgrams of FNC-RED or FNC (0, 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml, from bottom to top) binding to and dissociation from murine TLR4/MD-2 immobilized on the sensor chip are depicted in RU after subtracting the control signal. All data are representative of at least three independent experiments.

Article Snippet: Recombinant mouse LBP and recombinant mouse CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Transfection, Structural Proteomics, Expressing, Control, Concentration Assay, Flow Cytometry, Cell Culture, Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

doi: 10.1074/jbc.M117.791780

Figure Lengend Snippet: FNC-RED-induced TLR4/MD-2 activation does not require LBP and CD14. A, indicated concentrations of rmLBP and rmCD14 were pre-incubated with lipid A or FNC-RED in the absence of FBS for 30 min. Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were then treated with the recombinant protein/ligand mixtures in the absence of FBS for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Dashed lines depict those cultured with medium alone. Open histograms depict those stimulated with lipid A or FNC-RED. Filled histograms depict those stimulated with the recombinant protein/ligand mixtures. Black and gray values depict MFI of GFP expression in cultured cells stimulated with lipid A or FNC-RED and medium, lipid A, or FNC-RED with indicated recombinant proteins, respectively. B, Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were treated with anti-mouse CD14 mAb (10 μg/ml) or isotype control antibodies (Ct. IgG2b) (10 μg/ml) for 30 min and subsequently stimulated with the indicated concentration of lipid A or FNC-RED in the presence of FBS (10% v/v) for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Dashed lines depict those cultured with medium alone. Open histograms depict those stimulated with lipid A or FNC-RED. Filled histograms depict those stimulated with lipid A or FNC-RED in the presence of anti-mouse CD14 mAb or Ct. IgG2b. Black and gray values depict MFI of GFP expression in cultured cells stimulated with lipid A or FNC-RED and lipid A or FNC-RED with indicated Abs, respectively. Data are representative of at least three independent experiments.

Article Snippet: Recombinant mouse LBP and recombinant mouse CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Expressing, Recombinant, Flow Cytometry, Cell Culture, Control, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

doi: 10.1074/jbc.M117.791780

Figure Lengend Snippet: FNC-RED stimulation is impaired in CD14-dependent TLR4/MD-2 activation. A, Ba/F3 cells expressing murine TLR4-FLAG, murine TLR4-GFP, murine MD-2-FLAG, and murine CD14 were stimulated with medium alone, LPS (0.03 or 1 μg/ml), or FNC-RED (50 μg/ml) for 60 min. The cultured cells were then subjected to immunoprecipitation with anti-GFP and immunoblotting (IB) with anti-FLAG or anti-GFP as described under the “Experimental procedures.” B, Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 were stimulated with medium (Med) alone or the indicated concentration of lipid A or FNC-RED in the presence of FBS (10% v/v) for 1 h. The cells were harvested and stained with control antibodies Ct. Ab or anti-mouse TLR4/MD-2 mAb (clone MTS510). Open and filled histograms depict those stained with Ct. Ab and MTS510, respectively. Percentages of TLR4/MD-2-positive cells are depicted in each histogram. C, BM-cDCs from WT mice were stimulated with medium alone or the indicated concentration of lipid A or FNC-RED in the presence of FBS (10% v/v) for 1 or 18 h. The cells were harvested and stained with Ct. Ab or MTS510. Open and filled histograms depict those stained with Ct. Ab and MTS510, respectively. Percentages of TLR4/MD-2-positive cells were depicted in each histogram. D, BM-cDCs from WT and Cd14−/− mice were stimulated with medium alone or indicated concentration of lipid A or FNC-RED in the absence of FBS for 1 or 18 h. The cells were harvested and stained with Ct. Ab or MTS510. Open and filled histograms depict those stained with Ct. Ab and MTS510, respectively. Percentages of TLR4/MD-2-positive cells were depicted in each histogram. All data are representative of at least three independent experiments.

Article Snippet: Recombinant mouse LBP and recombinant mouse CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Expressing, Cell Culture, Immunoprecipitation, Western Blot, Concentration Assay, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

doi: 10.1074/jbc.M117.791780

Figure Lengend Snippet: Derivative of FNC-RED with a phosphate group activates human TLR4/MD-2. A, chemical structures of FNC-RED-P01 and FNC-P01. B, Ba/F3 cells expressing murine TLR4/MD-2 and murine CD14 (mTLR4/mMD-2/mCD14) or human TLR4/MD-2 and human CD14 (hTLR4/hMD-2/hCD14) were stimulated with the indicated concentration of FNC-RED-P01 or FNC-P01 for 18 h. The cells were harvested, and GFP expression was monitored by flow cytometry. Open and filled histograms depict those cultured with medium alone and FNC-RED-P01 or FNC-P01, respectively. Black and gray values depict MFI of GFP expression in cultured cells stimulated with medium alone and FNC-RED-P01 or FNC-P01, respectively. C, THP-1 cells were cultured as described under “Experimental procedures.” Total RNA was extracted, and expression levels of TNF-α, MCP-1, and IFN-β were measured by qRT-PCR. Data are shown as means ± S.D. N.S., not significant. §, p < 0.001 versus medium (Med.). Similar results were obtained in three independent experiments.

Article Snippet: Recombinant mouse LBP and recombinant mouse CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Cell Culture, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

doi: 10.1074/jbc.M117.791780

Figure Lengend Snippet: Schematic models of lipid A- or FNC-RED-stimulated TLR4/MD-2 activation. A, lipid A first binds to LBP and is transferred to the TLR4/MD-2 complex via either CD14 (membranous or soluble CD14)-dependent or CD14-independent manner. In CD14-dependent manner, lipid A induces tight dimerization of TLR4/MD-2 at the plasma membrane leading to TNF-α production via MyD88-dependent pathway. Furthermore, membranous CD14 (mCD14) is required for the internalization of TLR4/MD-2 to endosomes, where TRIF induces IFN-β production. In CD14-independent manner, TLR4/MD-2 is weakly dimerized by lipid A stimulation but can initiate MyD88- and TRIF-dependent pathways, leading to TNF-α production and CD86 up-regulation, respectively. B, FNC-RED stimulation induces CD14-independent but not CD14-dependent TLR4/MD-2 activation. Because CD14 or LBP is not involved in FNC-RED-stimulated activation, FNC-RED does not induce tight dimerization and internalization of TLR4/MD-2. Although the dimerization of TLR4/MD-2 is not detected in FNC-RED-stimulated Ba/F3 transfectant cells by immunoprecipitation assay, it may induce weak dimerization of TLR4/MD-2 at the plasma membrane to promote TNF-α production and CD86 up-regulation.

Article Snippet: Recombinant mouse LBP and recombinant mouse CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Transfection, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells

doi: 10.1084/jem.20011788

Figure Lengend Snippet: Transcriptional expression and immunostaining of m-IC cl2 cells for CD14. (A) Ribonuclease protection assay using specific antisense probes for murine mRNA encoding CD14 and GAPDH. Before RNA isolation, m-IC cl2 cells were exposed to 0.0, 0.1, or 10.0 μg/ml LPS for 12 h. RAW 264.7 cells were kept unstimulated. (B) Immunostaining for murine CD14 on m-IC cl2 cells. Cells were incubated for 12 h in the presence of various concentrations of LPS before fixation, as indicated. As control, immunostaining was performed on untreated cells by omitting the primary antibody. Immunostaining was detected using the horseradish peroxidase reaction and cells were counterstained with Mayer's hematoxylin. ×1,000. (C) Comparison of MIP-2 secretion in response to LPS stimulation by m-IC cl2 cells cultured in the presence or absence of serum. As control, 2 μg/ml recombinant CD14 was added before LPS stimulation.

Article Snippet: Cells were stimulated with LPS at a concentration of 10 ng/ml with or without the addition of 2 μg/ml recombinant CD14 (R&D Systems).

Techniques: Expressing, Immunostaining, Isolation, Incubation, Control, Comparison, Cell Culture, Recombinant

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Antibodies used for flow cytometric analysis.

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques:

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Antibodies used for capillary electrophoresis-based western blot analysis.

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques: Electrophoresis, Western Blot

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Antibody profile of the anti-CD147 antibody, h4 # 147D.

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques: Derivative Assay, Recombinant, Mutagenesis, Membrane, Sequencing

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Cell surface expression of CD147 and its binding partners on MIA PaCa-2cell-derived xenograft tumors after administration of h4 # 147D. (a) Antitumor effect of h4 # 147D in the mouse xenograft model. Five-week-old NOD SCID female mice were subcutaneously injected with MIA PaCa-2. After tumor volume reached 200 mm 3 , animals were randomized into groups of 6 mice and treated with either 1 or 3 mg/kg body weight h4 # 147D (i.v., single injection at day 0; 1 mg/kg blue arrow; 3 mg/kg green arrow). Vehicle-treated mice were used as a control group. Data show the mean and SEM of the estimated tumor volume. Asterisks indicate significant differences between each group connected by a line ( ∗∗∗ P < 0.001 and ∗ P < 0.05). (b) Protein expression analysis using flow cytometry. After treatment with h4 # 147D, expressions of CD147, CD44, integrin α 3, and integrin α 6 were detected using the antibodies listed in for cells prepared from tumors ( n = 3) on days 1, 3, 7, 10, and 14. Measured signals are shown as the percentage of the average signal from the control group. Graphs represent the mean per group ( n = 3 mice per group per time point), and error bars represent SEM. Asterisks indicate significant differences between the control group and antibody-treated groups ( ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05).

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques: Expressing, Binding Assay, Derivative Assay, Injection, Control, Flow Cytometry

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Protein expression of CD147, its binding partners, and downstream molecules in MIA PaCa-2 tumors after administration of the h4 # 147D antibody. Bar graphs show the mean ± SEM, n = 3 in all cases for the expression and luminescence signal ratio of CD147 (a), CD44 (b), integrin α 3 (c), integrin α 6 (d), MCT1 (e), phosphorylated-FAK, (Tyr397 (f) and Tyr925 (g)), total FAK (h), phosphorylated JNK (i), phosphorylated c-Jun (j), phosphorylated HSP27 (k), PAI-1 (l), and cleaved caspase-3 (m) to β -actin in tumor lysate samples from control tumors (isotype control-treated at 24 h or nontreated at 72 h and h4 # 147D-treated tumors (1 mg/kg or 3 mg/ml). Asterisks indicate significant differences between the isotype control-treated group at 24 h or nontreatment group (NT) as a control at 72 h and antibody-treated groups ( ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05).

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques: Expressing, Binding Assay, Control

Journal: Journal of Oncology

Article Title: Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals

doi: 10.1155/2022/3552793

Figure Lengend Snippet: Graphical representation of a possible mechanism of action (MoA) for h4 # 147D anti-tumor efficacy. Administration of h4 # 147D leads to decreased cell surface expression of CD147 and its binding partners (such as integrins, CD44, and MCTs) via inhibition of CD147 molecular chaperone functions. Decreased or displaced CD44 and integrins may induce inhibition of FAK and cytoskeleton stress-mediated activation of stress-responsive signals such as SMAD, JNK, and P38MAPK. These activated stress-responsive signals lead to caspase-3 activation and cell death. Inhibitory mechanisms for the multiple stress-responsivesignal-induced cell death are possible mechanisms of resistance to the h4 # 147 mechanism of action.

Article Snippet: Hybridomas producing the monoclonal antibody were generated from mice or rats after immunization with recombinant human CD147 protein (Creative BioMart, Shirley, NY, US) or CD147-expressing cells by the myeloma fusion method.

Techniques: Expressing, Binding Assay, Inhibition, Activation Assay

Journal: Nature Communications

Article Title: Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade

doi: 10.1038/ncomms14979

Figure Lengend Snippet: ( a ) Bright field (BF) images of co-cultures of EMT6 tumour cells (TC) (black arrow) with mMDSC (white arrows) or gMDSCs (red arrows) derived from 4T1 tumour-bearing mice. Morphological characteristic of EMT phenotype is visibly induced by the mMDSCs (top panel) which also show a strong affinity towards tumour cells. ( b – e ) mMDSC-induced EMT phenotype is assessed by enhanced expression of Vimentin and CK14. ( f ) Enhanced expressions of Vimentin and Twist induced by the mMDSCs from 4T1 tumour-bearing mice are verified by qPCR analyses. ( g – j ) mMDSCs from 4T1 tumour-bearing mice induce tumour cell invasion and CSC phenotype as shown by CD24-CD29 phenotype and tumour sphere forming assay. ( k , l ) In situ analyses of primary tumour and metastatic lesions by immunofluorescence staining reveal mMDSCs at the invasive edge of the primary tumour (TC, tumour center; TE, tumour edge) and gMDSCs around the pulmonary metastatic lesions in 4T1 tumour-bearing BALB/c mice (Met, metastasis; Inft, infiltrates). ( m ) Substantially higher number CD14-positive human mMDSCs were detected in metastatic human breast cancers compared to the indolent tumours. Results are presented as mean±s.d. ( n =3). Scale bar, 50 μm; * P <0.05, ** P <0.005; unpaired t -test.

Article Snippet: The following primary antibodies were used: Ly6C (#ab15627-Dilution 1/100, Abcam), Ly6G (#MAB1037-Dilution 1/100, R&D biosystem), Ki67 (#12202-Dilution 1/100, Cell Signaling), Vimentin (#550513 Dilution 1/100, BD Biosciences), CD14 (#10073-H08H Dilution 1/100, Sino biological).

Techniques: Derivative Assay, Expressing, In Situ, Immunofluorescence, Staining

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A, B Stimulated or unstimulated MDM were stained for classical macrophage markers (CD68, CD14) and M1 and M2 macrophage markers (CD163, CD80, CD86 and CD40) and analysed by FACS.

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Bone marrow derived macrophages (A, B) were stimulated with increasing concentrations of S-LPS in presence of 10 µg/mL of PIM 2 mimetic or deAcPIM 2 mimetic, or vehicle control, and TNF (A) and IL-12 p40 (B) were measured in supernatants after overnight incubation. Results are mean +/− SEM from n = 6 mice from three independent experiments. PIM analogues were titrated (C, D) in the presence of 0.1 µg/mL S-LPS and a 10 µg/mL dose was chosen as this concentration was sufficient for the active PIMs to strongly inhibit LPS-induced TNF (C) and IL-12 p40 (D) release without cytotoxicity. (E–J) HEK cells stably transfected with TLR4, MD2 and CD14 were incubated with PI (E), PIM 1 (F), isoPIM 1 (G), deAcPIM 2 mimetic (H) or PIM 2 mimetic (I) (10 µg/mL; dotted line) prior to incubation with biotinylated S-LPS (2,5 µg/mL) and streptavidin FITC (black line) or only streptavidin FITC (grey histogram). Non transfected HEK cells were incubated with biotinylated S-LPS as a control (J). Results are from one experiment representative of three independent experiments. (K) Percentage of S-LPS-binding to HEK-MTC cells in presence of vehicle, PI, isoPIM 1 , deAcPIM 2 mimetic or PIM 2 mimetic. Results are the mean +/− SD from three independent experiments. (L) Human IL-8 was measured in the supernatant after overnight S-LPS stimulation of HEK-MTC cells. Results are mean +/− SD from triplicates, from one experiment representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle; θ, p<0.05; θθθ, p<0.001 indicate significant differences between PIM 1 or isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic and deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 µg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Derivative Assay, Incubation, Analogues, Concentration Assay, Stable Transfection, Transfection, Binding Assay

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 mice (A, C) or CD14 KO mice (B, D) were incubated with synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL) or control vehicle prior to stimulation with Malp2 (30 ng/mL; A, B) or Pam 3 CSK 4 (Pam 3 ; 0.5 µg/mL; C, D). TNF release was measured in supernatants after overnight incubation. Results are mean +/− SD from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; †, p<0.05; †††, p<0.001, indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 µg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Macrophages from C57Bl/6 or CD14 KO mice were stimulated with increasing concentrations of S-LPS (A, B) or Re-LPS (C, D). TNF (A, C) and IL-12 p40 (B, D) concentrations were measured in the supernatants after overnight incubation. Results are mean +/− SEM from n = 4 mice from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences between C57Bl/6 and CD14 KO.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 µg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

doi: 10.1371/journal.pone.0024631

Figure Lengend Snippet: Concentrations of TNF (A–C) and IL-12 p40 (D–F) in supernatants of wild type (A, B, D, E) or CD14-deficient (C, F) macrophages stimulated overnight with 100 ng/mL of S-LPS (A, D) or Re-LPS (B, C, E, F) in the presence of synthetic PI, isoPIM 1 , deAcPIM 2 mimetic, PIM 2 mimetic (10 µg/mL), or vehicle. Results are mean +/− SD from n = 4 mice from two independent experiments representative of three independent experiments. ND: not detected. **, p<0.01; ***, p<0.001 versus vehicle. θθ, p<0.01; θθθ, p<0.001 indicate significant differences between isoPIM 1 versus PI as control; ††, p<0.01; †††, p<0.001 indicate significant differences between PIM 2 mimetic versus deAcPIM 2 mimetic as control.

Article Snippet: Soluble recombinant mouse CD14 was coated overnight at 4°C (5 µg/mL on Nunc 96-well plates; R&D systems, Abingdon, UK) and non specific binding saturated with 2% BSA in PBS for 1 hr at 37°C.

Techniques: