Mission sirna transfection reagent

UMRC3-LUC-GFP were cultured as described in the Supplementary Materials. 30% confluent cells were subcultured to a new plate/dish a day before transfection. siRNAs were transfected into cells according to the manufacturer’s instructions (Millipore Sigma-Aldrich). Briefly, 1 nM siRNA was transfected using 1 μl MISSION siRNA Transfection Reagent (Millipore Sigma-Aldrich, S1482). All siRNAs were bought from Millipore Sigma-Aldrich. Three different siRNA pairs targeting the ASCT2 sequence were used. Sequences were ASCT2-1 (5’-GUCAGCAGCCUUUCGCUCA-3’, 5’-UGAGCGAAAGGCUGCUGAC-3’), ASCT2-2 (5’-CCAAGCACAUCAGCCGUUU-3’, 5’-AAACGGCUGAUGUGCUUGG-3’), and ASCT2-3 (5’-GAGGAUGUGGGUUUACUCU-3’, 5’-AGAGUAAACCCACAUCCUC-3’). A negative control contained only transfection reagent. Cells were harvested 24 h post-transfection. ASCT2-3 was used for ASCT2 knockdown in the [¹⁸F]FGln cellular uptake experiment. [¹⁸F]FGln uptake and Western blot experiments were performed 48 h after transfection.

Different pre-designed MISSION^® siRNAs (Sigma, UK) were tested for effects on reducing Brn-3b expression in SKOV3 cells by transfecting different amounts of siRNA (6, 30 and 60 pmol), using the MISSION^® siRNA Transfection Reagent, in accordance with the manufacturer’s protocol. This was compared with cells transfected with an unrelated non-silencing siRNA that was used as a control. Western blot analysis of total cellular proteins taken at different times (72–96 hours) after transfection was used to determine effectiveness of knockdown. In addition, to study the effects of chemotherapeutic drugs after reduction of Brn-3b, transfected cells were treated with different drug combinations for 24 hours before further analysis (e.g. MTT assays).

HO-1 siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Briefly, 3 × 10⁵ cells were harvested in a 6 well-plate and transfected with 30 nM of specific siRNA, or the transfection reagent as a control. The MISSION^® siRNA Transfection Reagent was used according to manufacturer’s instructions (Sigma-Aldrich). After transfection, the plate was incubated at 37 °C for 24 h. The transfected cells were then transferred to a 25-cm² flask and maintained for 72 h to allow for cell recovery.

Rap1 siRNA (90 nM, SASI_Hs01_00040403; Sigma-Aldrich), R-Ras siRNAs (90 nM, siRNA mix, EHU0225113; Sigma-Aldrich), or negative control siRNA (90 nM, SIC-001; Sigma-Aldrich) were transfected into cells according to the manufacturer's instructions. Briefly, cells were incubated in cell culture inserts for four days (medium changed every second day). The medium was changed to 10% FCS Endothelial Cell Growth Medium 2 (C-22211; PromoCell) without heparin (10% EGM-2/heparin(−)). Then, a mixture of MISSION siRNA Transfection Reagent (S1452; Sigma-Aldrich) and siRNA was pre-incubated for 10 min, and then transfected. Two days later (6th day after starting cultivation), the medium was changed to 10% EGM-2/heparin(−) and siRNA transfection, and BSA and TAGE treatment were performed.

siRNA knock down and the cell proliferation assay were performed as previously described [8 (link)]. To knockdown AMPK, a possible target of metformin, we used AMPKɑ1/2 siRNA (h) (sc–45312, Santa Cruz, Santa Cruz, USA) and Control siRNA (sc–36869, Santa Cruz). For transfection, LNCaP cells were plated at a density of 1×10⁵ cells /well in 6-well plates and transfected with 10 nM AMPKɑ1/2 siRNA (h) or Control siRNA using MISSION^® siRNA Transfection Reagent (Sigma-Aldrich). Twenty-four hours after transfection, cells were subjected to the cell proliferation assay. Briefly, cells were detached and re-plated in 12-well tissue culture plates in complete media with or without 0.1 mM metformin. Three days after the treatment, cells were collected and counted using a hemocytometer.

siRNA oligonucleotides were synthesised from Genepharma, ShangHai, China. siRNA sequences targeting ATG5 [42 (link)]: siRNA 1#, 5′-GGUUUGGACGAAUUCCAACUUGUUU-3′; siRNA 2#, 5′-GAUCACAAGCAACUCUGGAUGGGAU-3′ and siRNA 3#, 5′-GCCAUCAAUCGGAAACUCAUGGAAU-3′; negative control RNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. The siRNAs were transfected into HeLa cells using Mission siRNA Transfection Reagent (Cat# S1452, Sigma-Aldrich, St Louis, MO, USA).