Journal: Biological Procedures Online

Article Title: Construction of a bacterial autoinducer detection system in mammalian cells

doi: 10.1251/bpo98

Figure Lengend Snippet: COS-1 cells expressing either of the indicated QscR-based proteins were fixed and QscR proteins were detected using an antiserum directed against the Myc epitope as described in materials and methods. The nuclei of the cells were stained with, 6’-diamidino-2-phenylindone (DAPI, blue image in central panel). The left and center images were merged to detect overlap between the signals.

Article Snippet: The monkey kidney COS-1 cell line was cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD) supplemented with 10% Cosmic Calf Serum (HyClone, Logan, UT).

Techniques: Expressing, Staining

Journal: Biological Procedures Online

Article Title: Construction of a bacterial autoinducer detection system in mammalian cells

doi: 10.1251/bpo98

Figure Lengend Snippet: Mammalian expression vectors encoding the indicated RhlR-based chimeric proteins were co-transfected into COS-1 cells with either the (lasBOX 1)2-Luc or (lasBOX 2)2-Luc reporter plasmids and luciferase values were measured. The activity detected in cells transfected with the (lasBOX 1)2-Luc plasmid alone was set to 1.0 and the activity measured for the other transfections were calculated relative to this value. All data points were performed in triplicate and data representative of three repetitions are shown.

Article Snippet: The monkey kidney COS-1 cell line was cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD) supplemented with 10% Cosmic Calf Serum (HyClone, Logan, UT).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

Journal: Biological Procedures Online

Article Title: Construction of a bacterial autoinducer detection system in mammalian cells

doi: 10.1251/bpo98

Figure Lengend Snippet: COS-1 cells were cotransfected with expression vectors encoding the indicated RhlR-based chimeric proteins and the (lasBOX 1)2-Luc reporter plasmid in the absence or presence of C4-HSL for 40 hours. Luciferase values were measured and expressed relative to the activity of the reporter plasmid alone.

Article Snippet: The monkey kidney COS-1 cell line was cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD) supplemented with 10% Cosmic Calf Serum (HyClone, Logan, UT).

Techniques: Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Biological Procedures Online

Article Title: Construction of a bacterial autoinducer detection system in mammalian cells

doi: 10.1251/bpo98

Figure Lengend Snippet: COS-1 cells were cotransfected with a mammalian expression plasmid encoding T3N-LasR and the (lasBOX 1)2-Luc reporter plasmid. The cells were incubated in the absence or presence of either 250μM 3O-C12-HSL or 750μM C12-HSL for three different time periods and luciferase activities were measured. The data are presented relative to the activity of the reporter plasmid alone. The highest activity of T3N-LasR was observed after 8 hours incubation with either 3O-C12-HSL or C12-HSL.

Article Snippet: The monkey kidney COS-1 cell line was cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD) supplemented with 10% Cosmic Calf Serum (HyClone, Logan, UT).

Techniques: Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: British Journal of Cancer

Article Title: Interleukin-1 α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

doi: 10.1038/bjc.2012.429

Figure Lengend Snippet: Fibroblasts as target for irritants and alarmins. ( A ) Monolayers of primary cultures of N-Fb, passage 4, were treated for 15 min with the irritants or cytokines listed at the bottom. The changes in the phosphorylation of the signalling proteins indicated on the right were determined by western blotting. ( B, C ) N-Fb were treated for 24 h as indicated and then were exposed for 15 min to CSFE-stained neutrophils. Adherent neutrophils were imaged by CSFE-fluorescence ( B , bottom panel, and C ) and the change in the expression of ICAM-1 as well as the neutrophil-associated haptoglobin determined by western blotting ( B , upper panels). ( D ) separate cultures of N-Fb were treated with the cytokines indicated at the bottom and analysed for the 15-min response to activate NFkB and p38 and for the 24-h response to induce ICAM-1 and binding of CSFE-stained neutrophils. ( E ) Baseline and IL-1 β -inducible IL-6 production by early ( P 3–10) and late ( P >10) passage culture of N-Fb and T-Fb were quantified by bioassay and Luminex immunobead-binding assays. Box plots compile data from 8 to 24 independent culture sets.

Article Snippet: Confluent Fb cultures in 48-well plates (∼1 × 10 5 cells per well) were treated with DMEM containing 10% fetal bovine serum (150 μ l per well) alone or containing either 1/3 to 1/10 diluted Ep CM; 10 ng ml −1 of recombinant IL-1 β or IL-1 α (17 kD; Peprotech, Rocky Hill, NJ, USA); IL-1F9 or IL-33 (R&D); or COS-1 cell-derived IL-1 α (17 kD+31 kD forms; Genetics Institute/Wyeth Research, Boston, MA, USA); 100 ng ml −1 oncostatin M (OSM) ( Chattopadhyay et al , 2007 ); 100 ng ml −1 hyper-IL-6 ( Rakemann et al , 1999 ); 1 μ g ml −1 LPS (Sigma-Aldrich); or 10 μ g ml −1 heat-killed Salmonella .

Techniques: Western Blot, Staining, Fluorescence, Expressing, Binding Assay, Luminex

Journal: British Journal of Cancer

Article Title: Interleukin-1 α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

doi: 10.1038/bjc.2012.429

Figure Lengend Snippet: Size separation of proteins in CM of PDT-treated T-Ep. Duplicate cultures of T-Ep, passage 3, in a 10-cm-diameter culture dish were treated either as light control or with 25 nℳ HPPH-PDT. Conditioned medium 2 and 24 h post PDT were concentrated and subjected to chromatography on a G-150 Sephadex column. Proteins in each fraction were analysed for the indicated proteins ( A, upper panel), FSA in the coupled Fb-H-35 cell assay ( B ) and for induced neutrophil binding to the treated Fb ( C ). Detection of the FSA activity by chromatographed COS-1 cell-derived IL-1 α is included in ( B and C ). The centrifuged homogenate of untreated T-Ep from one 10-cm dish was similarly size fractionated and the entire fractions were analysed for IL-1 α protein by western blotting ( A, lower panel). The elution positions of the molecular size markers (in kDa) are indicated at the top of the column elution profile.

Article Snippet: Confluent Fb cultures in 48-well plates (∼1 × 10 5 cells per well) were treated with DMEM containing 10% fetal bovine serum (150 μ l per well) alone or containing either 1/3 to 1/10 diluted Ep CM; 10 ng ml −1 of recombinant IL-1 β or IL-1 α (17 kD; Peprotech, Rocky Hill, NJ, USA); IL-1F9 or IL-33 (R&D); or COS-1 cell-derived IL-1 α (17 kD+31 kD forms; Genetics Institute/Wyeth Research, Boston, MA, USA); 100 ng ml −1 oncostatin M (OSM) ( Chattopadhyay et al , 2007 ); 100 ng ml −1 hyper-IL-6 ( Rakemann et al , 1999 ); 1 μ g ml −1 LPS (Sigma-Aldrich); or 10 μ g ml −1 heat-killed Salmonella .

Techniques: Chromatography, Binding Assay, Activity Assay, Derivative Assay, Western Blot

Journal: British Journal of Cancer

Article Title: Interleukin-1 α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

doi: 10.1038/bjc.2012.429

Figure Lengend Snippet: Identification of IL-1 α and IL-1 β as Ep-derived FSA and alarmin. Conditioned medium of control Ep cultures ( Ep-PDT ) and PDT-treaded Ep cultures ( Ep+PDT ), whole-cell homogenate of control Ep and Sephadex G-150-purified 17 kD FSA were reacted with antibodies against IL-1 α or IL-1 β as indicated and then tested for the ability to activate signalling ( A ), to enhance IL-6 that induces GFP expression in H-35 cells ( B ) and to increase neutrophil binding ( C ).

Article Snippet: Confluent Fb cultures in 48-well plates (∼1 × 10 5 cells per well) were treated with DMEM containing 10% fetal bovine serum (150 μ l per well) alone or containing either 1/3 to 1/10 diluted Ep CM; 10 ng ml −1 of recombinant IL-1 β or IL-1 α (17 kD; Peprotech, Rocky Hill, NJ, USA); IL-1F9 or IL-33 (R&D); or COS-1 cell-derived IL-1 α (17 kD+31 kD forms; Genetics Institute/Wyeth Research, Boston, MA, USA); 100 ng ml −1 oncostatin M (OSM) ( Chattopadhyay et al , 2007 ); 100 ng ml −1 hyper-IL-6 ( Rakemann et al , 1999 ); 1 μ g ml −1 LPS (Sigma-Aldrich); or 10 μ g ml −1 heat-killed Salmonella .

Techniques: Derivative Assay, Purification, Expressing, Binding Assay

Journal: British Journal of Cancer

Article Title: Interleukin-1 α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

doi: 10.1038/bjc.2012.429

Figure Lengend Snippet: Genes inducible by Ep-derived alarmins in normal pulmonary fibroblasts

Article Snippet: Confluent Fb cultures in 48-well plates (∼1 × 10 5 cells per well) were treated with DMEM containing 10% fetal bovine serum (150 μ l per well) alone or containing either 1/3 to 1/10 diluted Ep CM; 10 ng ml −1 of recombinant IL-1 β or IL-1 α (17 kD; Peprotech, Rocky Hill, NJ, USA); IL-1F9 or IL-33 (R&D); or COS-1 cell-derived IL-1 α (17 kD+31 kD forms; Genetics Institute/Wyeth Research, Boston, MA, USA); 100 ng ml −1 oncostatin M (OSM) ( Chattopadhyay et al , 2007 ); 100 ng ml −1 hyper-IL-6 ( Rakemann et al , 1999 ); 1 μ g ml −1 LPS (Sigma-Aldrich); or 10 μ g ml −1 heat-killed Salmonella .

Techniques:

Journal: British Journal of Cancer

Article Title: Interleukin-1 α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

doi: 10.1038/bjc.2012.429

Figure Lengend Snippet: Enhanced cell damage and release of FSA by cell surface-restricted photoreaction. A , 100 nℳ HPPH-Gal was bound to the cell surface of T-Ep, passage 3, during 30-min incubation at 4° C followed by internalisation and lysosomal deposition during a 24-h incubation at 37° C. Fluorescent micrographs of the cultures at × 100 magnification are shown. ( B, C) Replicate sets T-Ep cultures in 6-well dishes were reacted with the indicated concentrations of HPPH-Gal restricted to cell surface or to be internalised. The cell-associated fluorescence was quantified ( B , open circles) and the cells in all sets were treated on ice with 665 nm light. One set of cultures was immediately lysed and analysed by western blotting to the extent of STAT3 crosslinking and loss of EGFR ( C ). The other set was cultured for an additional 24 h to determine survival rate ( B , closed circle). ( D , E ) Duplicate set of T-Ep, passage 5, were subjected to HPPH-Gal binding to cell surface or lysosomal accumulation at the indicated HPPH-Gal dose. The photoreaction was carried out at 0° C and the cultures were incubated for an additional 24 h. Conditioned media collected after 2 h and 24 h were analysed for FSA on N-Fb cultures ( D ). Equivalent aliquots of cell lysates and conditioned media were analysed by western blotting for the indicated DAMPs ( E ). ( F , G ) Replicate cultures of T-Ep in 6-well plates were subjected to high-dose mitochondrial HPPH-PDT ( F ) or cell-surface HPPH-Gal PDT ( G ) with light treatment carried out on ice. After 4-h post-PDT incubation, the adherent cells, combined with cellular debris recovered by centrifugation of CM, were homogenised. Aliquots of the cell homogenates and cell-free-CM were analysed for the amount of IL-1 activity per culture.

Article Snippet: Confluent Fb cultures in 48-well plates (∼1 × 10 5 cells per well) were treated with DMEM containing 10% fetal bovine serum (150 μ l per well) alone or containing either 1/3 to 1/10 diluted Ep CM; 10 ng ml −1 of recombinant IL-1 β or IL-1 α (17 kD; Peprotech, Rocky Hill, NJ, USA); IL-1F9 or IL-33 (R&D); or COS-1 cell-derived IL-1 α (17 kD+31 kD forms; Genetics Institute/Wyeth Research, Boston, MA, USA); 100 ng ml −1 oncostatin M (OSM) ( Chattopadhyay et al , 2007 ); 100 ng ml −1 hyper-IL-6 ( Rakemann et al , 1999 ); 1 μ g ml −1 LPS (Sigma-Aldrich); or 10 μ g ml −1 heat-killed Salmonella .

Techniques: Incubation, Fluorescence, Western Blot, Cell Culture, Binding Assay, Centrifugation, Activity Assay