Stable and Transient Cell Line Generation

Cell lines (ATCC) were grown in DMEM (Life Technologies) with 10% FBS (Hyclone). MCF-7 and T47D cells stably expressing shRNA targeting PTEN or mismatch control (shMM) were described previously [41 (link)]. Generation of other stable Cell lines is described in Supplementary Methods. In transient transfection experiments, cells were transfected with pcDNA plasmid encoding myc-PREX1-HA (from Atanasio Pandiella, CSIC-Universidad de Salamanca) or FLAG-HA control (Addgene) plus Lipofectamine 2000. Cells were transfected with siRNA targeting P-REX1 or AllStars Negative Control (Qiagen, Dharmacon) plus Lipofectamine RNAiMAX (Life Technologies). Cells were treated with IGF-1 (PeproTech), heregulin (HRG), BKM-120, GDC-0941, MK-2206 (Selleckchem), or EHT1864 (Tocris Bioscience) as indicated in figures.

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Dillon L.M., Bean J.R., Yang W., Shee K., Symonds L.K., Balko J.M., McDonald W.H., Liu S., Gonzalez-Angulo A.M., Mills G.B., Arteaga C.L, & Miller T.W. (2014). P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT, and MEK/ERK signaling in breast cancer. Oncogene, 34(30), 3968-3976.

Publication 2014

Cell lines DMEM FLAG-HA control Lipofectamine 2000 Lipofectamine RNAiMAX IGF-1 BKM-120 GDC-0941 MK-2206 EHT1864

Cell lines Cells Eht1864 Gdc 0941 Heregulin Igf 1 Lipofectamine Lipofectamine 2000 Mk 2206 Plasmid Pten Shrna Sirna Transfection Transient

Corresponding Organization : Dartmouth College

Other organizations : Dartmouth–Hitchcock Medical Center, Vanderbilt University, The University of Texas MD Anderson Cancer Center, Breast Cancer Research Foundation

Top 5 similar protocols

Variable analysis

independent variables

ShRNA targeting PTEN
Mismatch control (shMM)
PcDNA plasmid encoding myc-PREX1-HA
FLAG-HA control
SiRNA targeting P-REX1
AllStars Negative Control
IGF-1
Heregulin (HRG)
BKM-120
GDC-0941
MK-2206
EHT1864

dependent variables

Cell growth and proliferation

control variables

DMEM (Life Technologies) with 10% FBS (Hyclone)
Lipofectamine 2000
Lipofectamine RNAiMAX (Life Technologies)

positive controls

MCF-7 and T47D cells stably expressing shRNA targeting PTEN
PcDNA plasmid encoding myc-PREX1-HA
SiRNA targeting P-REX1

negative controls

MCF-7 and T47D cells stably expressing mismatch control (shMM)
FLAG-HA control
AllStars Negative Control

Annotations

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