Manipulation of Progesterone Receptor Signaling

T47D-Y (PR-null) and MCF7 (PR-low) (19 (link)) cells were stably transfected with pSG5 or pSG5-PR-B and pSV-Neo or pIRES or pIRES-PR-B using FuGENE reagent (ThermoFisher) to generate multiple vector matched clones. Cells were cultured as previously described (12 (link), 19 (link)). Additional MCF7 cells were obtained from ATCC (PR-A/B+), cultured as indicated, and stably infected with retrovirus expressing LXSN, LXSN-wtPELP1, or LXSN-ΔNLS PELP1 (34 (link)). MCF7C4-12 (PR-null), MCF7L (PR-A/B+), BT474 (PR-A/B+), and MCF7 1GX (PR-A/B+) cells were cultured as described (12 (link), 26 (link), 36 (link), 50 (link)), MCF7L PR knockdown cells were generated using lentivirus shRNAs targeting PR (5 sequences) or GFP in pLKO (ThermoFisher).
Cells were treated with estradiol (1nM), tamoxifen (100nM), and ICI 182,780 (1uM) obtained from Sigma-Aldrich (St. Louis, MO); LY-294002 (10uM), U0126 (10uM), and PP2 (10uM) obtained from Calbiochem (Darmstadt, Germany); R5020 (10nM) purchased from NEN Life Science Products (Boston, MA); and IGF1 (5nM) from GroPep Bioreagents (Thebarton SA, Aus).

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Daniel A.R., Gaviglio A.L., Knutson T.P., Ostrander J.H., D’Assoro A.B., Ravindranathan P., Peng Y., Raj G.V., Yee D, & Lange C.A. (2014). Progesterone receptor-B enhances estrogen responsiveness of breast cancer cells via scaffolding PELP1- and estrogen receptor-containing transcription complexes. Oncogene, 34(4), 506-515.

Publication 2014

FuGENE reagent estradiol tamoxifen ICI 182,780

B cells Cells Clones Estradiol Fugene Ici 182 780 Igf1 Lentivirus Ly 294002 Mcf7 Pelp1 R5020 Retrovirus Shrnas Tamoxifen U0126 Vector

Corresponding Organization :

Other organizations : University of Minnesota, Twin Cities Orthopedics, Mayo Clinic, The University of Texas Southwestern Medical Center

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Variable analysis

independent variables

Transfection of cells with pSG5 or pSG5-PR-B and pSV-Neo or pIRES or pIRES-PR-B using FuGENE reagent
Stable infection of MCF7 cells with retrovirus expressing LXSN, LXSN-wtPELP1, or LXSN-ΔNLS PELP1
Treatment with estradiol (1nM), tamoxifen (100nM), ICI 182,780 (1uM), LY-294002 (10uM), U0126 (10uM), PP2 (10uM), R5020 (10nM), and IGF1 (5nM)

dependent variables

Measured outcomes not explicitly mentioned

control variables

Cell culture conditions as previously described (12, 19, 26, 36, 50)

positive controls

MCF7 cells obtained from ATCC (PR-A/B+)

negative controls

T47D-Y (PR-null) and MCF7 (PR-low) cells
MCF7C4-12 (PR-null) cells

Annotations

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