Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Proposed mechanism of SP600125-mediated suppression of nicotine-related AAA in the C57BL/6J mouse model. AngII, angiotensin II; JNK, c-Jun N-terminal kinase; MCP-1, monocyte chemoattractant protein-1; RANTES, regulated-on-activation, normal T-cell expressed and secreted; MMP, matrix metalloproteinase; ECM, extracellular matrix.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Activation Assay

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: SP600125 suppresses the protein expression of chemokines in aortic tissue. (a) Representative transverse sections of mouse aortic tissue obtained after transient perfusion with nicotine plus AngII, with or without SP600125. MCP-1 and RANTES proteins were undetectable in normal aorta but were abundant in nicotine plus AngII-induced AAA tissues (a1 and a2), where they appear to be expressed in the medial and outer smooth muscle cells. SP600125 inhibited the protein expression of MCP-1 and RANTES in AAA lesions (a3). (c, d) Representative western blots for MCP-1 and RANTES. The band optical density (OD) values (mean ± SD) of MCP-1 and RANTES were evaluated using ImageJ. GAPDH was used as an internal control and results are from independent triplicate experiments. Chemokine expression in the serum as detected by ELISA (e). ∗∗ P < 0.01 versus coadministration.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

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

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Expression of MCP-1 and RANTES under a concentration gradient of nicotine in MOVAS cells. Cellular mRNA expression levels of MCP-1 and RANTES (a, b) and the levels of secreted MCP-1 and RANTES in the supernatant (c, d) are shown. MCP-1 and RANTES expression as well as secretion was induced by nicotine in a dose-dependent fashion. The strongest expression was observed for 0.5 ng/mL and 5 ng/mL nicotine. Data are from independent triplicate experiments. ∗ P < 0.05 and ∗∗ P < 0.01 versus control; # P < 0.05 and ## P < 0.01 versus the group treated with 5 ng/mL nicotine.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Expressing, Concentration Assay, Control

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Influence of SP600125 on nicotine-induced expression of MCP-1 and RANTES in MOVAS cells. Cellular mRNA expression levels of MCP-1 and RANTES (a, b) and the levels of secreted MCP-1 and RANTES in the supernatant (c, d) are shown. Nicotine at 5 ng/mL could significantly upregulate the cellular mRNA expression as well as secretion of MCP-1 and RANTES, while SP600125 eliminated this effect. Data are from independent triplicate experiments. ∗ P < 0.05 and ∗∗ P < 0.01 versus control; # P < 0.05 and ## P < 0.01 versus the group treated with 5 ng/mL nicotine.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Expressing, Control

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Higher serum levels of GDF3 are related to poor outcomes of septic patients. ( A ) Scatter plots of serum GDF3 levels on admission of healthy donors and septic patients. ( B ) AUC discriminating sepsis from healthy controls. ( C , D ) Correlation between serum GDF3 levels and ( C ) APACHE II score and ( D ) SOFA score at admission of ICU. ( E ) Serum GDF3 levels in different groups sorted by survivor and non-survivor. ( F ) AUROC predicting 28-day mortality: GDF3 (AUC 0.770), Lac (AUC 0.767), CRP (AUC 0.632), PCT (AUC 0.677), SAA (AUC 0.724). (*, p < 0.05 vs. healthy donors; #, p < 0.05 vs. survivor).

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques:

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: The dynamic expression of GDF3 in mice and macrophages treated with endotoxin. ( A ) mRNA levels of GDF3 in different tissues of mice, n = 4. GAPDH gene expression was used as the internal control. ( B ) Serum GDF3 levels are detected at indicated time points in mice after LPS (10mg/kg) injection (*, p < 0.05; n = 4). mRNA levels of GDF3 were determined in ( C ) whole blood and ( D ) spleens of LPS-injected mice (*, p < 0.05; n = 6). ( E ) LPS-dose response and ( F ) time course of GDF3 expression in BMDMs, where indicated, ( E ) total RNAs were collected at 24 h after LPS exposure (*, p < 0.05) and ( F ) BMDMs were pre-treated with LPS (10 ng/mL) (*, p < 0.05; n = 4). Similar results were obtained in other two independent experiments.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Expressing, Gene Expression, Control, Injection

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Recombinant GDF3 protein suppresses pro-inflammatory cytokine production in LPS-treated macrophages. ( A – D ) BMDMs treated with the indicated doses of recombinant GDF3 (rGDF3) and ( E – H ) RAW264.7 macrophages treated with rGDF3 (50 ng/mL) for 2 h, followed by addition of LPS (10 ng/mL) for 24 h. Culture supernatants were measured for ( A , E ) TNF-α, ( B , F ) IL-6, ( C , G ) IL-1β and ( D , H ) MCP-1 by ELISA (*, p < 0.05; n = 7–9 wells). Similar results were obtained in another separated experiments.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Recombinant GDF3 protein reduces macrophage M1 and increases M2 polarization in LPS-treated BMDMs. BMDMs were treated with rGDF3 (50 ng/mL) for 2 h, then added LPS (10 ng/mL) for 6 h. mRNA levels of ( A ) iNOS and ( B ) Arg-1 were evaluated by RT-PCR. The mRNA levels were normalized to 18S mRNA levels and expressed as fold versus BSA group (*, p < 0.05 vs. Ctl; #, p < 0.05 vs. LPS; n = 6). ( C , D ) BMDMs were treated with rGDF3 (50 ng/mL) for 2 h, followed by LPS (10 ng/mL) exposure for 12 h. These BMDMs were stained with antibodies to ( C ) CD38 and ( D ) CD206 for flow cytometry analysis. Quantification results for ( E ) CD38 positive macrophages and ( F ) CD206 positive macrophages. (*, p < 0.05 vs. BSA; #, p < 0.05 vs. LPS; n = 3). Similar results were obtained in other two separated experiments.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Recombinant, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Recombinant GDF3 protein attenuates inflammatory response and mortality in endotoxin-induced septic mice. WT mice injected with rGDF3 protein (I.P., 10μg/kg BW) 12 h prior to LPS injection (I.P., 10mg/kg BW), 12 h later serum was collected for measuring ( A ) TNF-α, ( B ) IL-6, and ( C ) MCP-1 levels (*, p < 0.05 vs. Ctl; #, p < 0.05 vs. LPS + B; n = 5). ( D ) Kaplan-Meier survival curves were generated for LPS-mice treated with or without rGDF3 (*, p < 0.05; n = 12).

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Recombinant, Injection, Generated

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Administration of rGDF3 to LPS-mice improves cardiac function and promotes cardiac macrophage M2 phenotype. WT mice received recombinant GDF3 protein (I.P., 10μg/kg BW) 12 h prior to LPS injection (I.P., 10mg/kg BW), 12 h later cardiac function was measured by echocardiograph. ( A ) Representative M-mode echocardiography recordings for three groups, and ( B ) left ventricular ejection fraction (EF %), ( C ) fractional shortening (FS %) and ( D ) left ventricular internal dimension at end-systolic (LVIDs) were calculated (*, p < 0.05 vs. Ctl; #, p < 0.05 vs. LPS+B; n = 5). Cardiac cells were stained with antibodies to ( E ) MHC-II, ( G ) CD206, ( I ) F4/80 plus CD11b, ( K ) Ly6C for flow cytometry analysis and their respective quantification results are shown in ( F , H , J , L ). (*, p < 0.05; n = 4). Similar results were obtained in other two independent experiments.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Recombinant, Injection, Staining, Flow Cytometry

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: GDF3-mediated macrophage M2 phenotype is associated with the activation of Samd2/3 and inhibition of NLRP3 inflammasome. ( A ) Representative immuno-blots and quantification results for ( B ) p-Samd2, ( C ) p-Smad3 and ( D ) NLRP3. GAPDH was used as a loading control for total protein (*, p < 0.05 vs. Ctl cells, #, p < 0.05 vs. LPS treated cells; n = 3). ( E – G ) RAW264.7 macrophages were pre-treated with SB431542 or DMSO (10μM) for 0.5 h, then incubated with rGDF3 (50 ng/mL) or BSA for 1 h, followed by stimulation with LPS (10 ng/mL) for 12 h. Supernatants were collected to measure ( E ) TNF-α, ( F ) IL-6, and ( G ) IL-1β levels (*, p < 0.05 vs. Ctl cells; #, p < 0.05 vs. LPS-treated cells; n = 4). The mRNA levels of ( H ) iNOS and ( I ) Arg-1 in these macrophages treated as above were measured by RT-PCR. The fold changes were normalized to 18S (*, p < 0.05 vs. LPS-cells; n = 4). Data shown were representative of three independent experiments.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques: Activation Assay, Inhibition, Western Blot, Control, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

doi: 10.3390/cells9010120

Figure Lengend Snippet: Scheme depicting GDF3-drived protection against endotoxin-induced inflammation, cardiac dysfunction and mortality. Exogenous GDF3 binds to ALK4/5/7 where activates Smad2/3 and inhibits NLRP3 inflammasome, consequently suppresses macrophage pro-inflammatory phenotype (M1), leading to reduced inflammation, cardiac dysfunction, and mortality in endotoxin-induced septic mice.

Article Snippet: Recombinant mouse GDF3 protein (R&D Systems, Minneapolis, MN, USA) was added with BSA as a carrier protein to enhance protein stability and increase shelf-life.

Techniques:

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: A small population of ACC cells H295R overexpresses Ptch1 at the plasma membrane . ( A ) H295R were labeled with an anti-Ptch1 antibody directed against the extracellular loop and cells presenting Ptch1 at their plasma membrane (H295R-PM-Ptc+ AF594+ cells) were sorted. AF594+ in blue represents the percentage of cells with Ptch1 at the cell surface (H295R-PM-Ptc+ cells). ( B ) Surface labeling of Ptch1 using anti-Ptch1 antibody directed against the extracellular loop of Ptch1 (Alexa 594 in red) on nonpermeabilized parental H295R and H295R-PM-Ptc+ cells. Nuclei were stained with DAPI (in blue). The histogram represents the mean ± SEM of Alexa 594 fluorescence intensity per cell (****: p -value < 0.00005 ( p -value = 2 × 10 −36 )).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Clinical Proteomics, Membrane, Labeling, Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: H295R-PM-Ptc+ cells are more resistant to chemotherapy than parental cells . ( A ) Doxorubicin (dxr) cytotoxicity. H295R and H295R-PM-Ptc+ cells were treated for 48 h with increasing concentrations of dxr before cell viability measure. ( B ) Doxorubicin IC50 of H295R-PM-Ptc+ and H295R parental cells in the absence or the presence of 10 μM of the Ptch1 efflux inhibitor methiothepin. ( C ) H295R-PM-Ptc+ cells accumulate less doxorubicin than parental H295R cells. Cells on coverslips were incubated with 2 μM dxr for 15, 30, 60, 180 and 240 min and immediately fixed with PFA. Dxr fluorescence was acquired using a filter for Alexa 594 and quantified using ImageJ software. About 100 cells (from three wells) were scored per condition per experiment. All data presented are the mean ± SEM of at least 3 independent experiments. Significance is attained at p -value < 0.05 (*), (**** p < 0.00005).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Incubation, Fluorescence, Software

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Differentially expressed genes (DEG) between H295R-PM-Ptc+ and parental H295R cells selected for their role in cancer. Genes overexpressed are indicated in red and genes underexpressed are in blue.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Activation Assay, Expressing, Inhibition, Gene Expression, Migration, Marker, Biomarker Discovery

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Composition of active modules containing one or more of the identified genes of interest listed in Table 1 (in bold) with genes upregulated in red and genes downregulated in blue, representative enrichment and role of differentially expressed genes (DEGs) in cancers.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Migration, Cell Differentiation, Activation Assay, Membrane, Activity Assay