Transfections
DU145 cells were seeded at 1.2 × 105 cells per well in a 12-well plates for the PTEN-3’UTR luciferase assays. 24 hr later cells were transfected using Lipofectamine 2000 (Life Technologies, cat# 11668500) according to manufacturer’s instructions. Briefly, 2 µl Lipofectamine 2000 was mixed with Opti-MEM so the total volume was 100 µl and incubated for 10 min at RT. 100 ng of psiCHECK-2+PTEN3’UTR (Addgene, plasmid# 50936; RRID:Addgene_50936) and 100 pmol of siRNA or 1 µg of 3’UTR plasmid were brought to a total volume of 100 µl with Opti-MEM. The two solutions were gently mixed and incubated for an additional 20 min at RT. The transfection mixture was added to the appropriate well of cells and incubated at 37°C in a humidified atmosphere at 5% CO2. After 4 hr, growth medium was replaced. Cells were incubated for 72 hr at 37°C in a humidified atmosphere at 5% CO2 until harvested.
DU145, wild-type HCT116, and HCT116 DICEREx5 cells were seeded at 1.3 × 105 cells per well in 12-well plates and grown overnight for the Western blot and cell growth assays. The next day cells were transfected using Dharmafect 1 (Thermo Fisher Scientific, cat# T200104) according to manufacturer’s instructions with 100 nM siRNA. The transfection mixture was added to the appropriate well of cells and incubated at 37°C in a humidified atmosphere at 5% CO2. For the Western blot assay, growth medium was replaced after 4 hr. Cells were incubated for 72 hr at 37°C in a humidified atmosphere at 5% CO2 until harvested. For the Crystal violet proliferation assay, cells were trypsinized after 8 hr and seeded in new plates.
Transfection efficiency was determined with a siGLO RISC-Free transfection control condition 48 hr after transfection by fluorescence microscopy, which was confirmed to be >90% for all experiments reported. siRNA reagents: siGLO RISC-free (Dharmacon, cat# D-001600–01), siGenome non-targeting control 2 (Dharmacon, cat# D-001210–02), siGenome SERINC1 (Dharmacon, cat# M-010725–00), siGenome ZNF460 (Dharmacon, cat# M-032012–01), siGenome VAPA (Dharmacon, cat# M-021382–01), siGenome CNOT6L (Dharmacon, cat# M-016411–01), and siGenome PTEN (Dharmacon, cat# M-003023–02). 3’UTR plasmids: pcDNA-SERINC1-3’UTR, pcDNA-VAPA-3’UTR1, pcDNA-VAPA-3’UTR2, pcDNA-CNOT6L-3’UTR1, pcDNA-CNOT6L-3’UTR2, pCMV-MCS-PTEN-3’UTR (shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center).