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Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches.

Licciulli S, Luise C, Zanardi A, Giorgetti L, Viale G, Lanfrancone L, Carbone R, Alcalay M - BMC Cell Biol. (2010)

Bottom Line: It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes.Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Oncology, Istituto Europeo di Oncologia, Via Adamello 16, 20139, Milan, Italy.

ABSTRACT

Background: Pirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.

Results: We performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array).

Conclusions: The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

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Related in: MedlinePlus

ICA antibody spotting scheme. Each cell line was plated in one well of a 4-well slide; each antibody was spotted in 7 replicas on each well (see Methods for details)
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Figure 2: ICA antibody spotting scheme. Each cell line was plated in one well of a 4-well slide; each antibody was spotted in 7 replicas on each well (see Methods for details)

Mentions: Cells were seeded on gelatin-coated multiwell-slides at comparable densities (50-60% confluence), and different antibodies were simultaneously hybridized to multiple spots on the array (Figure 2). An affinity-purified polyclonal anti-PIR antibody was used to assess PIR expression. A polyclonal antibody recognizing the C-terminus of the ShcA adaptor protein was used as control for exclusive cytoplasmic localization, while anti-PIR total serum and pre-immune serum were included in the experiment as further controls. Signals were revealed with Cy3 labelled goat anti-mouse or anti-rabbit secondary antibodies, and nuclei were stained with Diamino-2-phenylindole (DAPI, Figure 3a, b). Image analysis was performed using a specific freeware software (CellProfiler) [20] to obtain fluorescence values corresponding to nuclear and cytoplasmic staining for each single cell. The average values of fluorescence for each antibody were then calculated.


Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches.

Licciulli S, Luise C, Zanardi A, Giorgetti L, Viale G, Lanfrancone L, Carbone R, Alcalay M - BMC Cell Biol. (2010)

ICA antibody spotting scheme. Each cell line was plated in one well of a 4-well slide; each antibody was spotted in 7 replicas on each well (see Methods for details)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823719&req=5

Figure 2: ICA antibody spotting scheme. Each cell line was plated in one well of a 4-well slide; each antibody was spotted in 7 replicas on each well (see Methods for details)
Mentions: Cells were seeded on gelatin-coated multiwell-slides at comparable densities (50-60% confluence), and different antibodies were simultaneously hybridized to multiple spots on the array (Figure 2). An affinity-purified polyclonal anti-PIR antibody was used to assess PIR expression. A polyclonal antibody recognizing the C-terminus of the ShcA adaptor protein was used as control for exclusive cytoplasmic localization, while anti-PIR total serum and pre-immune serum were included in the experiment as further controls. Signals were revealed with Cy3 labelled goat anti-mouse or anti-rabbit secondary antibodies, and nuclei were stained with Diamino-2-phenylindole (DAPI, Figure 3a, b). Image analysis was performed using a specific freeware software (CellProfiler) [20] to obtain fluorescence values corresponding to nuclear and cytoplasmic staining for each single cell. The average values of fluorescence for each antibody were then calculated.

Bottom Line: It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes.Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Oncology, Istituto Europeo di Oncologia, Via Adamello 16, 20139, Milan, Italy.

ABSTRACT

Background: Pirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.

Results: We performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array).

Conclusions: The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

Show MeSH
Related in: MedlinePlus