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: 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

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 1. Antitumor effect of intratumoral IFN-α gene transfer. (a) Growth of tumors injected with Ad-mIFN. Tumor volumes were measured at indicated days following the intratumoral injection of Ad-mIFN (n = 6) or Ad-AP (n = 8). Relative tumor volumes compared with those at day10 were presented. Data are shown as means ± standard deviation (s.d.). (b) ELISpot assay of IFN-γ-producing cells in response to stimulation of CT26 cells. Twenty-two days after tumor inoculation, splenocytes were isolated from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3), and were cultured with CT26 or syngeneic lymphocytes. Data are presented as means ± s.d. (c) Intracellular cytokine staining of IFN-γ-producing cells in response to CT26 cells. The splenocytes from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3) at day 22 were incubated with CT26 cells and stained by anti-mouse IFN-γ antibody. The activated cell fractions were analyzed by staining with anti-mouse CD8 antibody. Representative FACS plots (right panel) are shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Standard Deviation, Enzyme-linked Immunospot, Isolation, Cell Culture, Staining, Incubation

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 2. Intratumoral IFN-α gene transfer reduced the frequency of Tregs in tumors. (a) Frequency of CD4+Foxp3+ Tregs per CD4+ T cells in tumors. Tumors injected with viruses were harvested at days 16, 22 and 28, and processed into single-cell suspension. The percentage of CD4+

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Suspension

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 3. Intratumoral IL-6 concentration was significantly increased by IFN-α gene transfer. (a) IL-6 concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at days 16, 22 and 28, and IL-6 concentration was measured by ELISA. (b) Relationship between IL-6 and IFN-α concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at day 16, and the concentrations of IL-6 and IFN-α were compared by ELISA. (c) IL-6 production from tumor CD11c+ cells. The CD11c+ and CD11c −cells were isolated from tumors injected with Ad-mIFN (n = 2) or Ad-AP (n = 2) at day 16, and 5 × 104 cells were plated in 96-well plates. After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA. (d) IL-6 production from splenic CD11c+ cells in response to a recombinant IFN-α protein. The CD11c+ and CD11c−cells isolated from naïve splenocytes, and 5 × 104 of CT26 cells were cultured in 96-well plates with depicted concentration of recombinant mouse IFN-α (Miltenyi Biotech). After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA (n = 2 for each group).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 4. IL-6 receptor blockade suppressed IFN-α-mediated Treg reduction in tumors. (a) Schema of experiment. The 1000 μg of the monoclonal anti-IL-6 receptor antibody was intraperitoneally injected into the mice at days 7, 14 and 21 after tumor inoculation. Ad-mIFN or Ad-AP was injected once at day 10 after inoculation. (b) Frequency of CD4+Foxp3+ cells per CD4+ T cells in tumors treated with IL-6R ab. Tumors were harvested at day 22, and CD4+ T cells and CD4+Foxp3+ Tregs were analyzed by flow cytometry (n = 5 for the group of Ad-AP i.t. +IL-6R ab i.p., n = 4 for the other groups). (c) Ratio of CD8+ T cells to CD4+Foxp3+ Tregs in tumors. Frequency of CD8+ T cells within whole tumor cells (left panel). Frequency of CD4+Foxp3+ Tregs within whole tumor cells (middle panel). The number of CD8+ T cells was compared with that of CD4+Foxp3+ Tregs in tumors at day 16 (right panel) (n = 4 for the group of Ad-mIFN i.t.+IL-6R ab i.p., n = 6 for the other groups). IL-6R ab, anti-IL-6 receptor antibody; i.t., intratumoral injection; i.p., intraperitoneal administration; TDLNs, tumor-draining lymph nodes.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Cytometry

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 5. IL-6 receptor blockade partially attenuated IFN-α-mediated tumor growth suppression. (a) Growth of tumors treated with IL-6R ab. Tumor volumes in mice treated with the viruses and/or IL-6R ab were measured at the indicated days (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups). Relative tumor volumes compared with those at day 10 were presented. (b) ELISpot assay of IFN-γ- producing cells in mice treated with IL-6R ab. The splenocytes were isolated from mice as shown in Figure 4a at day 28, and the cells were cultured with CT26 or syngeneic splenocytes (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 6. Intratumoral IFN-α expression increased the number of Th17 cells in tumors. (a) Expression of RORγt and Foxp3 genes in tumors. The tumors injected with viruses were harvested at day 16 and were subjected to real-time PCR analysis (Ad-mIFN: n = 4, Ad-AP: n = 3). (b) Expression of IL-17 in tumors. The tumors were harvested at day 28, and subjected to RT-PCR for IL-17 expression (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 2 for the group of Ad-mIFN i.t.+PBS i.p., n = 3 for the group of Ad-mIFN i.t.+IL-6R ab i.p.). (c) Intracellular cytokine staining of IL-17A in CD4+ T cells. The tumors and tumor-draining lymph nodes were harvested at day 22, and IL-17A expressions were analyzed by flow cytometry (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 4 for the group of Ad-mIFN i.t.+PBS i.p., n = 4 for the group of Ad-AmIFN i.t.+IL-6R ab i.p.) (upper panel). Representative FACS plots of tumors (lower left panel) and tumor-draining lymph nodes (lower right panel) were shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry