Computational protocol: Stat3 regulates centrosome clustering in cancer cells via Stathmin/PLK1

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Protocol publication

[…] Recombinant GST-Stat3 protein was from Abnova (Taipei City, Taiwan), recombinant HIS-Stathmin protein was from Prospec (Burnaby, BC, Canada) and active recombinant PLK1 was from SignalChem. PLK1 Assay/Inhibitor Screening Kit (Cyclex, Nagano, Japan) was used following manufacturer's recommendations and the colorimetric assay in 96-well format was read on a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA, USA). A concentration of 1 mU PLK1 corresponds to an 8.9 nM concentration and therefore 1 × Stathmin corresponds to 8.9 nM concentration, 2 × Stathmin corresponds to 17.8 nM concentration and 10 × Stathmin corresponds to 89 nM concentration. Equimolar Stat3 was 17.8 nM concentration, corresponding to the 2 × Stathmin concentration used in the assay (). [...] Levels of γ-tubulin at the centrosome were measured by focusing on the middle (maximal size and intensity) of an individual centrosome, recording an image, then in Image J, placing a 1 μm circle at the centroid of the centrosome and measuring integrated pixel intensity. Background signal was subtracted and was measured by placing a circle in an area of the cell away from the centrosomes and measuring γ-tubulin integrated pixel intensity. Cells where individual centrosomes could not be distinguished were excluded from analysis.To plot γ-tubulin intensity, BoxPlotR was used ( Note that line scans of the type shown in were not used for quantifying γ -tubulin intensity at the centrosome.BT-549 cells were treated for 4 h with Stattic then fixed, stained and scored for the presence of long astral microtubules in mitotic cells. To ensure consistent results and to accurately determine which microtubules were astral microtubules, only cells with two centrosomes were scored and supernumerary centrosome cells were ignored for this assay. […]

Pipeline specifications

Software tools cycleX, BoxPlotR
Applications Miscellaneous, scRNA-seq analysis