Sigenome sirna smartpool reagents

Specific siGENOME siRNA SMARTpool® reagents against TPD54 as well as a negative control, siGENOME Non-Targeting siRNA, were purchased from Dharmacon Inc. (Lafayette, CO, USA). Detailed sequence information was provided in Additional file 1: Supplementary Materials and Methods. MCF-7 cells were transiently transfected with negative control or TPD54 siRNAs for 24 h using Lipofectamine RNAimax. Cells were then treated with metformin at different concentrations for 48 h.
shRNA plasmids (shTRC2 control and shTPD54 shRNA plasmids) were purchased from Sigma-Aldrich. MCF-7 cells were transfected with shRNA plasmids using the Lipofectamine 2000 transfection reagents. Transfected cells were then incubated in the presence of puromycin at 2 μg/mL for 2 weeks to generate stably transfected cells. Single colonies with low TPD54 expression were then selected for further downstream experiments.
TPD54 (NM_199359.2) was overexpressed in MCF-7 cells and patient-derived xenografts using lipofectamine 2000. Cells were treated as indicated after transfection for 1 day.

Specific siGENOME siRNA SMARTpool® reagents against a given gene, as well as a negative control, siGENOME Non-Targeting siRNA Pool #2, were purchased from Dharmacon Inc. (Lafayette, CO). Human pancreatic cancer SU86 and breast cancer MDA-MB-231 cell lines were used to perform the siRNA knockdown studies. The lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) was used for siRNA reverse or forward transfection. Specifically, cells were seeded into 96-well plates and were mixed with siRNA-complex consisting of 20–50 nM of specific siGENOME siRNA SMARTpool or non-targeting negative control (Dharmacon) and the lipofectamine RNAiMAX transfection reagent. The human leukemia cell lines, BDCM and THP-1, were transfected with electroporation using the Nucleofector System with 500 nM of specific or negative siRNA (Lonza Inc., Basel, Switzerland).

Cells were plated at 70% confluence in culture medium supplemented with 10% FBS, and were transfected with empty vector or CTSO plasmid (OriGene) using lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the vendor's protocol. Cells were collected for protein analysis 48 hours after transfection. In some experiments, 24 hours after transfection, cells were treated with 10 μM E-64, a cysteine proteases inhibitor, for additional 24 hours. Cells were then collected for protein analysis.
Specific siGENOME siRNA SMARTpool reagents against a given gene as well as a negative control, siGENOME Non-Targeting siRNA, were purchased from Dharmacon Inc. (Lafayette, CO, USA). Cells were transfected with control siRNA, and specific siRNAs (10nM) in 96-well plates or 12-well plates using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the vendor's protocol. For the purpose of cell growth assay, cells were plated in base medium supplemented with 5% charcoal stripped FBS for 24 hours, and then cultured in FBS-free RPMI 1640 media for another 24 hours before transfection. Different treatments were started 24 hours after transfection. For the purpose of testing gene expression level, cells were transfected with control siRNA and specific siRNAs (10nM) in 12-well plates using lipofectamine RNAiMAX for 48 hours.