Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: (A) The levels of IL-18 in the BAL fluid on day 1 after S. pneumoniae infection after RSV infection in various treatment groups (WT vs. Gas6–/–; WT vs. Axl–/–; IgG vs. Axl mAb treatment; hydroxypropyl methylcellulose vs. BGB324 treatment; PBS vs. rGas6 treatment; and control liposome vs. clodronate liposome treatment). The levels of IFN-γ, NO, and TNF-α in the BAL fluid from each group at day 1 after S. pneumoniae infection in the anti–IL-18 mAb-treated (B) and recombinant IL-18–treated (C) mice infected with S. pneumoniae and/or RSV. (D and E) IL-18 production in the alveolar macrophages isolated from WT and Gas6–/– mice on day 8 after RSV infection and stimulated with S. pneumoniae for 1 hour. (F) Activation of caspase-1 and degradation of IκB in the alveolar macrophages isolated from WT and Gas6–/– mice on day 8 after RSV infection and stimulated with S. pneumoniae for 3 hours (left panel). The ratios of cleaved caspase-1 (p10) and the IκB to GAPDH ratio are shown (right panel). The data are expressed as mean ± SEM; n = 4–6 (A and B), n = 2–3 (E and F). Representative results from 2 independent experiments are shown. The following statistical tests were used: 2-tailed Student’s t-test (A, for WT vs. Gas6–/–; WT vs. Axl–/–; IgG vs. Axl mAb treatment; hydroxypropyl methylcellulose vs. BGB324 treatment), and 1-way ANOVA (A, for PBS vs. rGas6 treatment; and control liposome vs. clodronate liposome treatment, B, C, and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The membrane was incubated with the rabbit anti-IκB (Abcam, ab32518, 1:500), anti–caspase-1 (Santa Cruz Biotechnology, sc56036, 1:100), or anti-GAPDH (control) antibodies (Abcam, ab8245, 1:1000) for 1 hour at room temperature.

Techniques: Infection, Recombinant, Isolation, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization

doi: 10.1172/JCI125505

Figure Lengend Snippet: Shortly after RSV infection, the epithelial cells and alveolar macrophages produce Gas6. The Gas6/Axl axis polarizes antibacterial (M0-like) macrophages into nonantibacterial (M2-like) macrophages that inhibit the activation of caspase-1/IL-18. The low levels of IL-18/IFN-γ in the macrophages and NK cells result in severe secondary pneumococcal infection.

Article Snippet: The membrane was incubated with the rabbit anti-IκB (Abcam, ab32518, 1:500), anti–caspase-1 (Santa Cruz Biotechnology, sc56036, 1:100), or anti-GAPDH (control) antibodies (Abcam, ab8245, 1:1000) for 1 hour at room temperature.

Techniques: Infection, Activation Assay

Journal: British Journal of Cancer

Article Title: NrCAM, a neuronal system cell-adhesion molecule, is induced in papillary thyroid carcinomas

doi: 10.1038/sj.bjc.6603915

Figure Lengend Snippet: Immunoblotting of neuron-glia-related cell-adhesion molecule (NrCAM). PTC and normal thyroid tissue protein extracts were subjected to western blot analysis with antibodies against NrCAM (ab24344, Abcam) or β -actin. ( A ) Semi-quantitative detection of NrCAM by western blotting. Increasing amounts of pooled protein extracts prepared from 10 normal and 10 papillary carcinoma thyroid tissue samples. Chemiluminescence signals (inset) were scanned and quantified as reported in Materials and Methods. Amounts of immunoreactive NrCAM, expressed as arbitrary units, are plotted as a function of the amount of membrane protein loaded in each lane. ( B ) Comparative analyses of NrCAM content of thyroid tumours and paired normal thyroid samples. Protein extracts (50 μ g) from each PTC and normal paired thyroid were subjected to western blot analysis and chemiluminescence detection. β -Actin was then detected on the same membranes and the signals used to normalise NrCAM values. Paired samples were analysed simultaneously. Data from five representative paired tumour/normal thyroid samples are shown. ( C ) Mean NrCAM protein content in a series of PTCs and normal thyroid (±s.e.m.) expressed as arbitrary units. T-papillary thyroid carcinoma; NT-paired normal thyroid tissue.

Article Snippet: Slices of 3–4 μ m were dewaxed, deparaffinised, rehydrated, and pretreated for antigen retrieval by exposure to 0.01% protease XXIV solution (Sigma, Steinheim, Germany) at 37°C for 10 min. After endogenous peroxidase activity quenching, and blocking of non-specific binding, sections were incubated with polyclonal goat anti-NrCAM antibody (1 : 200, clone sc-18960 – epitope mapped to an internal region of NrCAM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit polyclonal antibody (1 : 600, ab24344, directed against the extracellular NrCAM domain, aa 834–856; Abcam, Cambridge, UK) overnight at 4°C in a humid chamber, followed by incubation with LSAB+detection kit reagents (DAKO A/S Glostrup, Denmark), for 15 min at room temperature.

Techniques: Western Blot

Journal: Oncogene

Article Title: CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants.

doi: 10.1038/sj.onc.1207228

Figure Lengend Snippet: Figure 4 Overexpression of CD95L and of CD95 proteins after transient transfection. Inducible overexpression of CD95L (41 kDa) and CD95 (42 kDa, lower band) proteins is shown for three tetracycline-regulatable melanoma cell lines (SKM13-Tet-On, Bro-Tet-On and Mel2a-Tet-Off) after transient transfection; MeWo-Tet-On showed constitutive expression. Below the Western blots, induction by Doxy is indicated (7Doxy); proteins were induced by Doxy in SKM13-Tet-On and Bro-Tet-On and were repressed by Doxy in Mel2a-Tet-Off 0

Article Snippet: The following antibodies were used: CD95: rabbit polyclonal IgG (N-18, sc-714, Santa Cruz, Heidelberg, Germany, 1 : 1000); CD95L: rabbit polyclonal IgG (N-20, sc-834, Santa Cruz, 1 : 200); Bid: goat polyclonal IgG (N-19, sc-6539, Santa Cruz, 1 : 200); caspase-3: rabbit polyclonal IgG (Pharmingen, 556425; BD Biosciences, Heidelberg, Germany, 1 : 200); PARP: mouse monoclonal (Mol-Biol, Hamburg, Germany, 1 : 1000); cytochrome c: mouse monoclonal (Pharmingen, Heidelberg, Germany, 1 : 800); a caspase-8 antiserum was kindly supplied by K Schulze-Osthoff (Department for Molecular Medicine, Heinrich Heine University Düsseldorf, Germany).

Techniques: Over Expression, Transfection, Expressing, Western Blot

Journal: Oncogene

Article Title: CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants.

doi: 10.1038/sj.onc.1207228

Figure Lengend Snippet: Figure 6 Induction of apoptosis by conditional CD95L expression in stable cell clones. (a) Doxy inducibility of a 1650 nucleotide mRNA specific for CD95L is shown after Northern blotting in six SKM13-Tet-On cell clones stably transfected with pTRE-CD95L. Names of cell clones (503; 505) and treatment with Doxy (; þ ) are indicated. (b) Relative apoptotic rates were determined for these cell clones 48 h after induction with Doxy. Basic apoptotic rates determined for the cultures grown in absence of Doxy were individually set to 1. Each experiment consisted of triple values; means and standard deviations of three independent experiments are shown

Article Snippet: The following antibodies were used: CD95: rabbit polyclonal IgG (N-18, sc-714, Santa Cruz, Heidelberg, Germany, 1 : 1000); CD95L: rabbit polyclonal IgG (N-20, sc-834, Santa Cruz, 1 : 200); Bid: goat polyclonal IgG (N-19, sc-6539, Santa Cruz, 1 : 200); caspase-3: rabbit polyclonal IgG (Pharmingen, 556425; BD Biosciences, Heidelberg, Germany, 1 : 200); PARP: mouse monoclonal (Mol-Biol, Hamburg, Germany, 1 : 1000); cytochrome c: mouse monoclonal (Pharmingen, Heidelberg, Germany, 1 : 800); a caspase-8 antiserum was kindly supplied by K Schulze-Osthoff (Department for Molecular Medicine, Heinrich Heine University Düsseldorf, Germany).

Techniques: Expressing, Stable Transfection, Clone Assay, Northern Blot, Transfection

Journal: Oncogene

Article Title: CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants.

doi: 10.1038/sj.onc.1207228

Figure Lengend Snippet: Figure 7 Activation of death cascade after induction of CD95L. (a) Western blots for CD95L, procaspase-8, Bid and procaspase-3 are shown for SKM13-CD95L and the parental cell line SKM13-Tet-On. Proteins were extracted 24 and 48 h after induction with Doxy. Equal amounts of proteins (50 mg/lane) were loaded and equal transfer of proteins to the blotting membrane was confirmed by ponceau red staining. Inducibility of CD95L is seen in parallel to reduced protein levels of uncleaved Bid and of the proenzymes of caspase-3 and caspase-8 in SKM13-CD95L cells, whereas, the parental cells were not affected. Molecular weights of identified protein bands are given on the right side. (b) Cleavage products of caspase-8 (43, 41 and 18 kDa) were seen only in SKM13-CD95L cells after Doxy induction. (c) Increased caspase-3 enzyme activity is shown for SKM13-CD95L cells, 48 h after induction. Treatment with Doxy is indicated below; ‘I’ specifies addition of a caspase-3 inhibitor to the protein extract of induced cells, which blocked induction of caspase-3 activity. (d) Cytosolic and mitochondrial (Mito) extracts were obtained of SKM13-CD95L and SKM13-Tet-On cells treated with Doxy ( þ ) or left untreated (). Increased amounts of the proapoptotic factors cytochrome c and AIF were found in the cytosol of SKM13-CD95L after induction. b-actin and VDAC served as loading controls for cytosolic and mitochondrial fractions, respectively. Also, VDAC proved that cytosolic extracts were not contaminated with mitochondria

Article Snippet: The following antibodies were used: CD95: rabbit polyclonal IgG (N-18, sc-714, Santa Cruz, Heidelberg, Germany, 1 : 1000); CD95L: rabbit polyclonal IgG (N-20, sc-834, Santa Cruz, 1 : 200); Bid: goat polyclonal IgG (N-19, sc-6539, Santa Cruz, 1 : 200); caspase-3: rabbit polyclonal IgG (Pharmingen, 556425; BD Biosciences, Heidelberg, Germany, 1 : 200); PARP: mouse monoclonal (Mol-Biol, Hamburg, Germany, 1 : 1000); cytochrome c: mouse monoclonal (Pharmingen, Heidelberg, Germany, 1 : 800); a caspase-8 antiserum was kindly supplied by K Schulze-Osthoff (Department for Molecular Medicine, Heinrich Heine University Düsseldorf, Germany).

Techniques: Activation Assay, Western Blot, Membrane, Staining, Activity Assay

Journal: Oncogene

Article Title: CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants.

doi: 10.1038/sj.onc.1207228

Figure Lengend Snippet: Figure 9 Development of melanomas in nude mice is prevented by conditional CD95L expression in vivo. Tumor photographs were taken 7 weeks after subcutaneous injection of SKM13-CD95L cells into both flanks of nude mice. Large tumors had developed when mice were left untreated (a). When treatment was started 4 weeks after injection when already small tumors had developed, tumor growth was interrupted and delayed (b). Treatment with Doxy from the beginning of the experiment resulted in a complete block of tumor development (c). Each treatment group consisted of seven animals corresponding to 14 injection sites. The mice within one group showed similar response; one example of each group (abdomen) is shown here. The right graph shows the progression of the mean tumor size between days 30 and 61 after inoculation of SKM13-CD95L cells (means of each seven animals/14 tumors). Mice treated with Doxy after day 30 were characterized by tumor regression on the average for the first 3 weeks as well as by delayed tumor progression (b), as compared to untreated animals (a)

Article Snippet: The following antibodies were used: CD95: rabbit polyclonal IgG (N-18, sc-714, Santa Cruz, Heidelberg, Germany, 1 : 1000); CD95L: rabbit polyclonal IgG (N-20, sc-834, Santa Cruz, 1 : 200); Bid: goat polyclonal IgG (N-19, sc-6539, Santa Cruz, 1 : 200); caspase-3: rabbit polyclonal IgG (Pharmingen, 556425; BD Biosciences, Heidelberg, Germany, 1 : 200); PARP: mouse monoclonal (Mol-Biol, Hamburg, Germany, 1 : 1000); cytochrome c: mouse monoclonal (Pharmingen, Heidelberg, Germany, 1 : 800); a caspase-8 antiserum was kindly supplied by K Schulze-Osthoff (Department for Molecular Medicine, Heinrich Heine University Düsseldorf, Germany).

Techniques: Expressing, In Vivo, Injection, Blocking Assay

Journal:

Article Title: Fas and Fas Ligand Are Up-Regulated in Pulmonary Edema Fluid and Lung Tissue of Patients with Acute Lung Injury and the Acute Respiratory Distress Syndrome

doi:

Figure Lengend Snippet: Immunofluorescence co-localization of Fas and FasL protein expression in lung tissue sections from a patient who died from ALI or ARDS [identified as “(+) ARDS”; a] or from another patient who died without pulmonary disease [identified as “(−) ARDS”; b]. a: Cells that line the alveolar walls (arrow) and in the alveolar walls are orange, indicating co-localization of Fas (green) and FasL (red) proteins. b: Only a few cells are immunostained either green (Fas, arrow) or red (FasL, *) in the lung tissue section from a patient who died without pulmonary disease. Nuclei are blue because DAPI was used as the counterstain. Both panels are the same magnification.

Article Snippet: One set of 20 tissue sections each was incubated with either 1:250 dilution of anti-Fas mouse monoclonal anti-human antibody or 1:1000 dilution of anti-FasL polyclonal rabbit anti-human antibody (SC-8009 for anti-Fas and SC-834 for anti-FasL, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C.

Techniques: Immunofluorescence, Expressing