VDR Translocation Assay with Secosteroids

The effects of the new D₃ analogs on VDR translocation from the cytoplasm to the nucleus were tested using SKMEL-188 cells (33 (link)), stably transduced with pLenti-CMV-VDREGFP-pgk-puro (VDR and EGFP expressed as fusion protein) (21 (link), 22 (link)). Cells were treated with 10^–10-10^–7 M secosteroids overnight for 16 h followed by fixing with 4% paraformaldehyde (PFA). Fixed cells were mounted with fluorescent mounting media (Dako, Carpinteria, CA, USA) and analyzed with a fluorescence microscope. Translocation to the nucleus was determined by counting cells with a fluorescent nucleus and the results are presented as the percentage of the total cells that displayed nuclear staining, as described previously (21 (link)). The data were obtained from at least two separate experiments, with images taken randomly from at least six different fields and counted as described elsewhere (21 (link), 22 (link)).

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CHEN J., WANG J., KIM T.K., TIEU E.W., TANG E.K., LIN Z., KOVACIC D., MILLER D.D., POSTLETHWAITE A., TUCKEY R.C., SLOMINSKI A.T, & LI W. (2014). Novel Vitamin D Analogs as Potential Therapeutics: The Metabolism, Toxicity Profiling, and Antiproliferative Activity. Anticancer research, 34(5), 2153-2163.

Publication 2014

fluorescent mounting media

Cells Cytoplasm Fluorescence microscope Nucleus Paraformaldehyde Protein Secosteroids Translocation

Corresponding Organization :

Other organizations : University of Tennessee Health Science Center

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Variable analysis

independent variables

Concentration of secosteroids (10^-10 - 10^-7 M)

dependent variables

Percentage of cells with fluorescent nucleus (translocation of VDR-EGFP fusion protein to the nucleus)

control variables

SKMEL-188 cells stably transduced with pLenti-CMV-VDREGFP-pgk-puro (VDR and EGFP expressed as fusion protein)
Incubation time of 16 hours
Fixation with 4% paraformaldehyde (PFA)
Mounting of fixed cells with fluorescent mounting media

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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