Computational protocol: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

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Protocol publication

[…] SKOV-3 ovarian cancer cells were exponentially grown on sterilized cover glass (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and treated with 10−7 M ICI 182,780 for 30 min prior to the treatment of 10−7 M thyroxine or 10−9 M E2 for different time periods. The cells were immediately fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 min and then permeabilized in 0.1% Triton X-100 in PBS for 20 min. The cells on the slides were incubated with integrin αv antibody (1:200, Santa Cruz) or anti- ERα-phospho(S167)- antibody (1:200, Cell Signaling Technology) overnight at 4°C. Then cells were incubated with Alexa Fluor®-488 and Alexa Fluor®-647-conjugated secondary antibody (Abcam, Cambridge, United Kingdom) and mounted in EverBrite Hardset mounting medium with DAPI (Biotium, CA, USA). The fluorescent signals from integrin αv and phospho-ERα were recorded and analyzed with TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems, Wetzlar, Germany). The figures shown are representative of four fields for each experimental condition. Nuclei were defined by DAPI staining and nuclear fluorescence intensities were measured by ImageJ freeware (ImageJ, NIH, USA); data are shown as average intensity per cell. [...] Immunoblot and nucleotide densities were analyzed with IBM® SPSS® Statistics software (SPSS Inc., Chicago, IL, USA). Two tails student's t-test was conducted and considered significant at p-values < 0.05 (*, or †), 0.01 (**, or ††), 0.001 (***, or †††). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Ovarian Neoplasms
Chemicals Estradiol, Estrogens, Thyroxine