Characterization of PGRMC1 Mutants in Breast Cancer Cells

MCF7 and T47D cells were obtained from ATCC (Manassas, Virginia) and authenticated by Microsynth AG (Balgach, Switzerland) on October 7, 2016. Profiling of human cell lines was done using highly-polymorphic short tandem repeat loci (STRs). STR loci were amplified using the PowerPlex® 16 HS System (Promega, Madison, Wisconsin). Fragment analysis was done on an ABI3730xl (Thermo Fisher Scientific, Waltham, Massachusetts) and the resulting data were analyzed using GeneMarker HID software (Softgenetics, State College, Pennsylvania).
Cells were stably transfected with expression plasmid pcDNA3.1 containing hemagglutinin (HA)-tagged PGRMC1 wild-type or HA-tagged PGRMC1 mutants S57A, S181A and S57A/S181A, as described elsewhere [35 (link)]. MCF7 and T47D cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts), 100 units/ml penicillin/streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts) and 25 mM HEPES (Thermo Fisher Scientific, Waltham, Massachusetts) in a humidified incubator at 37°C with 5% CO₂.

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Willibald M., Bayer G., Stahlhut V., Poschmann G., Stühler K., Gierke B., Pawlak M., Seeger H., Mueck A.O., Niederacher D., Fehm T, & Neubauer H. (2017). Progesterone receptor membrane component 1 is phosphorylated upon progestin treatment in breast cancer cells. Oncotarget, 8(42), 72480-72493.

Publication 2017

PowerPlex® 16 HS System ABI3730xl GeneMarker HID software RPMI 1640 medium fetal bovine serum penicillin/streptomycin HEPES

Cell lines Cells Fetal bovine serum Hemagglutinin Hemagglutinin 1 Hepes Human Mcf7 Penicillin Pgrmc1 Plasmid Polymorphic Promega Short tandem repeat Streptomycin

Corresponding Organization :

Other organizations : Heinrich Heine University Düsseldorf, Düsseldorf University Hospital, Natural and Medical Sciences Institute, University of Tübingen

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Variable analysis

independent variables

Transfection of MCF7 and T47D cells with expression plasmid pcDNA3.1 containing HA-tagged PGRMC1 wild-type or HA-tagged PGRMC1 mutants S57A, S181A and S57A/S181A

dependent variables

Not explicitly mentioned

control variables

MCF7 and T47D cell lines obtained from ATCC and authenticated by Microsynth AG
MCF7 and T47D cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 25 mM HEPES in a humidified incubator at 37°C with 5% CO2

controls

Positive control: MCF7 and T47D cell lines obtained from ATCC and authenticated
Negative control: Not explicitly mentioned

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