Dharmafect 2 transfection reagent

After 18 h of culture, PHT cells were transfected with 100 nM si-RNAs targeting LAT1 and/or LAT2 transporters for 24 h using Dharmafect2 transfection reagent (Thermo Scientific, Rockford, IL), according to the manufacturer’s protocol. For both isoforms silencing efficiency was initially tested using 3 different si-RNA sequences (Sigma-Aldrich; for LAT1: SASI_Hs01_00103507, SASI_Hs01_00103508, SASI_Hs01_00103509; for LAT2: SASI_Hs01_00090978, SASI_Hs01_00090979, SASI_Hs01_00090980). Based on these pilot experiments (data not shown), SASI_Hs01_00103509 (LAT1) and SASI_Hs01_00090980 (LAT2) si-RNAs were chosen for the experiments described in this study. Equal concentration of non-targeting scrambled-siRNA (si-SCR, SIC001, Sigma-Aldrich) was used as a control. At 90 h of culture PHT cells were collected for Western blot and qRT-PCR analyses or used for transport assays. In each assay, the mean of controls (si-SCR) was assigned an arbitrary value of 1.0 and the data represents fold change from si-SCR.

On TARGET plus SMART-pool small interfering RNA (siRNA) sequences targeting Sp1 and non-targeting control were purchased from Dharmacon Research (Lafayette, CO, USA). Transfection was according to the manufacturer's instructions. HEp-2 and KB cells were seeded at 50%–60% confluence in 6-well plates and transfected transiently with 25 or 50 nM siRNA using DharmaFECT2 transfection reagent (Thermo Scientific, Lafayette, CO, USA). After transfected for 6 or 48 h, HEp-2 and KB cells were analyzed for apoptotic effects using western blots and DAPI staining.

Cells cultured in 6-well plates were treated with 19-mer siRNAs targeted against Ac45 (Thermo Scientific-Dharmacon SMARTpool M-021378-00, 25 nm), ATP6V0A1 (Thermo Scientific-Dharmacon SMARTpool M-017618-00, 100 nm), ATP6V0A3 (Thermo Scientific-Dharmacon SMARTpool M-012198-00, 100 nm), and ATP6V1A1 (Thermo Scientific Dharmacon SMARTpool L-017590-01, 50 nm). A control siRNA (Thermo Scientific-Dharmacon non-targeting pool D-001810-10) was also used at 100 nm. The four constituent siRNAs within each pool were also tested individually for effects on expression and for phenotypic effects. As an additional negative control, a representative siRNA from each pool was tested after custom synthesis (Thermo Scientific-Dharmacon) to include nucleotide changes as underlined: a₁, caacaucucaguaaugcua; a₃, cccucgcgcagcacaagua; Ac45, ccaccugucaacguaguuu).
The siRNAs were incubated with DharmaFECT-2 transfection reagent (Thermo Scientific) in serum-free medium for 45 min at room temperature before mixing 1:4 with complete medium (7% fetal bovine serum). Cells were treated with the siRNA mixture for 24 h before replacement with fresh complete medium. Where necessary for certain assays (invasion, microphysiometry), cells were transferred after this time and used within the subsequent 24 h.

HEK-293T cells were seeded into 6-well plates (3 × 10⁵ cells per well) for transfection on the following day. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with previously published plasmids [96 (link)], and then harvested 24 h post-transfection for immunoblotting. For siRNA transfections, 494H cells were seeded into 6-well plate (1.6 × 10⁴ cells per well) in antibiotic free media. The following day cells in each well were transfected with 50 nM of On-Targetplus non-targeting siRNA #2 (Thermo Fisher Scientific) or SMART pool: On-Targetplus mouse RIPK1 siRNA (Thermo Fisher Scientific) using 2 μl of DharmaFECT-2 transfection reagent (Thermo Fisher Scientific). Cells were incubated with transfection mixture for 8 h, before replacing media. Transfected cells were cultured for 48 h, and then harvested for immunoblotting and cell death assays.

Neuroblastoma cells were seeded in RPMI 1640 (GIBCO, Life Technologies) with 10% FCS and without antibiotics and transfected with the siRNA library against 131 cell cycle regulators (Dharmacon Cell Cycle ON-TARGETplus, Thermo Scientific) at a concentration of 100 nM using Dharmafect 2 transfection reagent (Thermo Scientific). The genes targeted by the siRNA library are listed in Additional file 1: Table S3. Transfections with siRNA against CCND1 and scrambled negative control siRNA were done using smart pools of siRNAs (Dharmacon, Thermo Scientific).

Dharmafect 2 transfection reagent (Thermo Scientific, Rockford, IL) and small interfering RNAs (siRNAs) (Sigma-Aldrich, St. Louis, MO), targeting raptor (100 nM; sense, 5′CAGUUCACCGCCAUCUACA), rictor (100 nM; sense, 5′CGAUCAUGGGCAGGUAUUA), DEPTOR (SASI_1297010-H/5582, 1297011-H) or Nedd4-2 (100 nM; 5′CCCUAUACAUUUAAGGACU) were used. Control cells were transfected with a non-coding scrambled sequence (100 nM; sense: 5′GAUCAUACGUGCGAUCAGATT). siRNAs were added to cultured PHT cells (~3.75 × 10⁶ cells/well in 6 well plate; ~7.5 × 10⁶ cells in 60 mm dish) after 18 h in culture, incubated for 24 h31 (link) and subsequently removed and replaced by fresh medium. At 90 hours in culture, efficiency of target silencing was determined at the protein and functional levels using Western blot.