Computational protocol: Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

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Protocol publication

[…] Individual transfected cells were three-dimensionally cropped into new files from the larger image sets described above for analysis. Cells expressing GFP or GFP-tagged IMP constructs were first identified using the Imaris software package (Bitplane, version 7). The Imaris batch processor mapped a “surface” around all cells within every image with GFP signal above a threshold level set from mock-transfected cell images. During the subsequent cell cropping, manual assessment was also performed, so cells were selected for analysis only if the entire cell nucleus was clearly defined and distinguishable within the image (x, y, and z); cells with overlapping nuclei or multinucleated cells were excluded. Rare cells containing cytoplasmic PSPC1 speckles were also excluded. To assess PSPC1 speckle number, size, and PSPC1 intensity within each cell, we used the Imaris batch processor to perform a “spots” analysis on each cell (individual image). Results were combined using the CSV Merge command, and the output files were manipulated using Excel (Microsoft, version 2007) and analyzed using SPSS software (IBM, version 17.0). […]

Pipeline specifications

Software tools Imaris, SPSS
Application Miscellaneous
Organisms Mus musculus