Transfection and Silencing Assays

Validated pools of FBXW7α-, MCL1-, PLK1-siRNA and non-targeting siRNA as negative control were obtained from GE Dharmacon (ON-TARGETplus SMART pools L-004264, L-004501, L-003290, and D-001810). Transfections were carried out using DharmaFECT 2 reagent (GE Dharmacon) according to manufacturer's instructions. All siRNA pools were used at 50 nM. Cells were subjected to different treatments 24 h after silencing. Transient transfections of pCMVHA and pCMVHA-FBXW7 plasmids [28 (link)] were carried out using FuGENE reagent (Promega) according to manufacturer's instructions. Cells were subjected to different treatments 24 h after transfection.

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Gasca J., Flores M.L., Giráldez S., Ruiz-Borrego M., Tortolero M., Romero F., Japón M.A, & Sáez C. (2016). Loss of FBXW7 and accumulation of MCL1 and PLK1 promote paclitaxel resistance in breast cancer. Oncotarget, 7(33), 52751-52765.

Publication 2016

DharmaFECT 2 reagent FuGENE reagent

Cells Fbxw7 Fugene Mcl1 Plasmids Plk1 Promega Sirna Transfection Transient

Corresponding Organization : Hospital Universitario Virgen del Rocío

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Variable analysis

independent variables

FBXW7α-siRNA
MCL1-siRNA
PLK1-siRNA
Non-targeting siRNA (negative control)
PCMVHA and pCMVHA-FBXW7 plasmid transfections

dependent variables

Cells subjected to different treatments 24 h after silencing
Cells subjected to different treatments 24 h after transfection

control variables

All siRNA pools were used at 50 nM
Transfections were carried out using DharmaFECT 2 reagent according to manufacturer's instructions
Transient transfections of pCMVHA and pCMVHA-FBXW7 plasmids were carried out using FuGENE reagent according to manufacturer's instructions

positive controls

Non-targeting siRNA (negative control)

negative controls

Non-targeting siRNA (negative control)

Annotations

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