In vitro 3H-FEAU uptake study

Quantitative evaluation of the uptake of radiotracer in vitro of TK in cells transient transfected with Lenti-CMV-DsRedm-tk and Lenti-STAT3-NF-κB-DsRedm-tk was measured by ³H-FEAU uptake assay as previously described ²⁴. In brief, MDA-MB-231 cells transfected with equal amount of Lenti-CMV-DsRedm-tk and Lenti-STAT3-NF-κB-DsRedm-tk plasmid with untransfected cells (control) were seeded in 12-well plates (1×10⁵ per well) in triplicate and incubated overnight, followed by 10 ng/mL TNF-α (R&D systems, cat.210-TA-005, Minneapolis, MN, USA) or DMSO treatment for 24 h. Culture medium was then replaced by 0.5 mL of medium containing 29-fluoro-29-deoxyarabinofuranosyl-5-ethyluracil (³H-FEAU) (7.4 kBq [0.2 μCi]), and incubated for 120 minutes at 37˚C with 5% CO₂. The medium and 0.5 mL of phosphate-buffered saline used to rinse the wells were then collected in scintillation vials. Cells were lysed by 75 μL of CytoBuster Protein Extraction Reagent (Novagen, US) and washed once with PBS. Then the cells were transferred to 1.5 mL tubes and centrifuged at 12,000 rpm for 1 minute. Supernatant aliquots (100 μL) were transferred to scintillation vials, and the remnant was used to determine total protein concentration. Thereafter, 5 mL of scintillation solution (Perkin-Elmer, Shelton, CT) was added to each vial and radioactivity of ³H-FEAU in samples was measured using a Packard Tri-Carb LS scintillation counter (Perkin-Elmer, Shelton, CT). Radioactivity concentration ratio of the cell pellet versus medium was calculated, and normalized by total protein concentration ([dpm/g of cells]/[dpm/g of medium]/total protein concentration). The ratios of accumulated radiotracer in cells represent the gene expression level of HSV-tk.