Journal:

Article Title: Autocrine growth inhibition by transforming growth factor ?-1 (TGF?-1) in human neuroendocrine tumour cells

doi:

Figure Lengend Snippet: Autocrine growth inhibition by transforming growth factor β (TGFβ) in neuroendocrine tumour (NET) cells. (A) BON and LCC-18 cells (1.5×104) were seeded and attached overnight. Cells were then incubated with 10 ng/ml TGFβ-1, a neutralising anti-TGFβ antibody (TGFβ-ab) or irrelevant control antibody (data not shown) at a final concentration of 20 μg/ml for three days. Cell number was then determined and expressed as a per cent of untreated controls. For determination of DNA synthesis, BON cells were incubated with TGFβ-1 or a neutralising anti-TGFβ antibody for 72 hours. For the last six hours, BrdU was added and DNA synthesis was then quantified by ELISA. Data represent mean (SEM) from three separate experiments, each conducted in triplicate (*p<0.05). (B) BON cells were stably transfected with a dn-TGFβR II-GFP expression construct or mock transfected with the GFP vector alone. Expression of the dn-TGFβR II-GFP fusion protein was verified by membrane localisation. (C) Transfected cell lines were analysed for growth as indicated under (A) to confirm the autoinhibitory action of endogenous TGFβ in BON cells. Shown are the mean (SEM) of three independent experiments, each performed in triplicate (*p<0.05).

Article Snippet: The following antibodies were purchased: anti-poly(ADP ribose) polymerase (PARP) antibody from Calbiochem/Oncogene Research (Bad Soden, Germany); c-myc and Smad 2/3 from BD PharMingen (Heidelberg, Germany); Smad 4/DPC4 and Smad3 from Upstate Biotechnology (Lake Placid, USA); proliferating cell nuclear antigen (PCNA), TGFβ-1 (does not cross react with TGFβ-2 and TGFβ-3), TGFβR I, and TGFβR II from Santa Cruz Biotechnology (Santa Cruz, USA), and secondary antibodies from Dianova (Hamburg, Germany).

Techniques: Inhibition, Incubation, Control, Concentration Assay, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Expressing, Construct, Plasmid Preparation, Membrane

Journal:

Article Title: Autocrine growth inhibition by transforming growth factor ?-1 (TGF?-1) in human neuroendocrine tumour cells

doi:

Figure Lengend Snippet: Expression of transforming growth factor β (TGFβ) signalling components in neuroendocrine tumour (NET) cell lines. (A) NET cell lines express TGFβ type I receptor (TGFβR I) and TGFβ type II receptor (TGFβR II) mRNA transcripts. RT-PCR analysis using primers specifically directed against TGFβR I, TGFβR II, and GAPDH was performed. Alternating lanes represent the results with (+) or without (−) prior reverse transcription to exclude genomic DNA contamination. A 100 bp DNA standard was used for size determination and the size of the amplicons are indicated. (B) Immunoblots using whole cell extracts (50 μg protein/lane) were performed to determine expression of TGFβR I, TGFβR II, Smad2, Smad3, and Smad4 protein. Immunodetectable proteins migrated at the expected molecular mass, as indicated. (C) TGFβ-1 protein is expressed and secreted in NET cell lines. Cells (2×104) were seeded overnight and cultured in serum free UltraCulture medium for three days. A TGFβ-1 specific ELISA was used to determine TGFβ-1 concentrations in the supernatants of the indicated cell lines and normalised to protein content/ml supernatant. Each experiment was performed in triplicate. (D) NET cell lines express TGFβ-2 and TGFβ-3 mRNA transcripts. RT-PCR analysis using primers specifically directed against TGFβ-2 and TGFβ-3 was performed. Alternating lanes represent the results with (+) or without (−) prior reverse transcription.

Article Snippet: The following antibodies were purchased: anti-poly(ADP ribose) polymerase (PARP) antibody from Calbiochem/Oncogene Research (Bad Soden, Germany); c-myc and Smad 2/3 from BD PharMingen (Heidelberg, Germany); Smad 4/DPC4 and Smad3 from Upstate Biotechnology (Lake Placid, USA); proliferating cell nuclear antigen (PCNA), TGFβ-1 (does not cross react with TGFβ-2 and TGFβ-3), TGFβR I, and TGFβR II from Santa Cruz Biotechnology (Santa Cruz, USA), and secondary antibodies from Dianova (Hamburg, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: Comparison of expression of IL-5 (a) and IL-1β (b) mRNAs using RT-PCR and Southern blot analysis in human ASM cells after 0-, 3-, 6-, and 24-hour exposure to human control serum and human atopic asthmatic serum. Expression of RPL7 mRNA was used to control for gel loading. The blots were probed with human specific IL-5, IL-1β, and RPL7 32P-labeled cDNA probes (see Methods). Whereas mRNA expression of IL-5 or IL-1β was unaltered in cells that were exposed to control serum, the mRNA signals for IL-5 and IL-1β were upregulated in cells that were exposed to atopic asthmatic serum. The temporal patterns of enhanced mRNA expression of these cytokines were characterized by an initial (at 3 hours) increased expression of IL-5 mRNA, followed (at 6 hours) by an increased mRNA expression of IL-1β. In contrast, mRNA expression of the constitutively expressed RPL7 gene was unaltered.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Labeling

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: Comparisons of the release of IL-5 and IL-1β proteins into the culture media of human ASM cells after 0-, 3-, 6-, and 24-hour exposure to human control serum (open symbols) and human atopic asthmatic serum (filled symbols). In contrast to control serum that had no appreciable effect, ASM cells exposed to human atopic asthmatic serum showed significantly increased levels of both IL-5 (filled circles) and IL-1β (filled squares) protein release into the cell culture media. Moreover, the temporal pattern for IL-5 release appeared relatively earlier (at 3 hours) than that for IL-1β release. Data represent mean ± SE values.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Cell Culture

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: (a) Comparison of human IL-1β mRNA expression examined using RT-PCR and Southern blot analysis in cultured human ASM cells after 0-, 3-, 6-, and 24-hour exposure to SFM alone (control) or IL-5. Relative to the unaltered constitutively expressed RPL7 mRNA signal, IL-1β mRNA expression was markedly upregulated at 3 and 6 hours in the IL-5–exposed ASM cells, whereas the IL-1β mRNA signal was virtually undetectable in the control cells. (b) Western blot analyses of human IL-1β expression in membrane/lysate samples isolated from cultured human ASM cells and tissues after 24-hour exposure to SFM alone and to IL-5. In contrast to control cells and tissues maintained in SFM alone, the cells and tissues treated with IL-5 exhibited markedly upregulated expression of IL-1β protein.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Cell Culture, Western Blot, Membrane, Isolation

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: Comparison of human IL-1β protein accumulation in the culture media of human ASM cells after 0-, 3-, 6-, and 24-hour exposure to SFM alone (control; open bars) or IL-5 (filled bars). Relative to unaltered release of IL-1β protein in control cells, ASM cells exposed to IL-5 exhibited significantly increased levels of IL-1β protein release into the cell culture media. Data represent mean ± SE values.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Comparison, Cell Culture

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: Comparison of contractile dose-response relationships to ACh in paired rabbit ASM segments after treatment with SFM alone (open circles) or with a maximum effective concentration of IL-5 (5 ng/mL) in the absence (filled circles) or presence (open squares) of IL-1ra. Relative to ASM exposed to SFM alone (control), the heightened constrictor responses to ACh in the IL-5–treated tissues were prevented by cotreatment of tissues with IL-1ra. Data represent mean ± SE values from 6 paired experiments.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Comparison, Concentration Assay

Journal:

Article Title: Autocrine interaction between IL-5 and IL-1? mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle

doi:

Figure Lengend Snippet: Comparison of relaxation dose-response relationships to isoproterenol in paired rabbit ASM segments half-maximally contracted with their respective ED50 doses of ACh after treatment with SFM alone (open circles) or with IL-5 in the absence (filled circles) or presence (open squares) of IL-1ra. Relative to ASM exposed to SFM alone (control), the attenuated relaxation responses to isoproterenol in the IL-5–treated tissues were prevented by cotreatment of tissues with IL-1ra. Data represent mean ± SE values from 6 paired experiments.

Article Snippet: The IL-1 receptor antagonist, anti-human IL-5 receptor and IL-4 neutralizing antibodies, IL-5 and IL-1β ELISA kits, mouse anti-human IL-1β primary antibody, and anti-mouse secondary antibody used in protein assay studies were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).

Techniques: Comparison