Three validated siRNA against TK1 and control siRNA (GAPDH) were purchased from Thermo Fisher Scientific (catalog # 4390824, siRNA IDs s14158, s14159, and s14160 for TK1, and catalog # 4390849 for GAPDH). siRNA was resuspended in nuclease-free water at 50 uM, aliquoted, and stored at −80˚C. For working siRNA concentrations, siRNA was diluted to 10 uM in nuclease-free water.

Two hundred-thousand A549 cells were plated in 6 well-plates overnight to allow for attachment, after which, the medium was removed, and cells were washed with DPBS and 2 mL serum-free RPMI 1640 was added to each well. Lipid-nucleic acid complexes of RNAiMAX (Thermo Fisher Scientific, Waltham, MA, catalog # 13778030) and siRNA were prepared according to the manufacturer’s instructions and added to the cells. 12 hours after transfection, medium and lipid-nucleic acid complexes were removed from cells and fresh complete RPMI 1640 medium was added to the cells. Cells were used at 60 hours post transfection, as recommended by manufacturer. (Cells treated with siRNA ID 14158 were named siRNA 58, those treated with siRNA ID 14159 were named siRNA 59, and those treated with siRNA ID 14160 were named siRNA 60).

Transfected A549 cells were rinsed with DPBS, treated with Accutase at 37°C for 5 - 10 mins to allow for detachment, and then rinsed with complete medium. Cells were pelleted and resuspended at 1 × 10⁶ cells/mL in Cell Staining Buffer (BioLegend, San Diego, CA) and 200 uL of cells were placed in individual microcentrifuge tubes and incubated with 5 uL of Fc block (Human TruStain FcX™, BioLegend, San Diego, CA) for 10 minutes at room temperature, followed by incubation with 1 ug of anti-TK1 antibody (ab91651, Abcam, Cambridge, United Kingdom) for 15 minutes on ice. Cells were then washed with 1 mLCell Staining Buffer, incubated with anti-rabbit AlexaFluor647 antibody (Thermo Fisher, Waltham, MA, cat # A27040) and incubated on ice in the dark for 15 minutes. Cells were washed 2X with Cell Staining Buffer and resuspended in Fluorescence-activated cell sorting (FACS) buffer. FACS buffer was made with phosphate-buffered saline (PBS), 2% calf serum (Thermo Fisher, Waltham, MA), 1 mM EDTA (Thermo Fisher, Waltham, MA, CAS 6381-92-6), and 0.1% sodium azide (Sigma Aldrich, St. Louis, MO, CAS 26628-22-8). We collected 1 × 10⁴ events per sample in a flow cytometer (Accuri C6 Plus, BD Biosicences, San Jose, CA) and data was analyzed using FlowJo software (FlowJo, Ashland, OR).

An xCELLigence RTCA DP instrument (Cat. 05469759001) was utilized to determine real time invasion and cell motility of A549 cells either expressing TK1 on their surface or after TK1 silencing. Invasion was assessed in 16 well CIM-Plates (Cat. 05665817001) (n = 4) composed of an upper and lower chamber, each containing 16 wells. The top wells were coated with a 1:40 Matrigel concentration and incubated for 4 hrs. Cells were plated in the top chamber at a concentration of 50,000 cells/well in serum-free RPMI in a total volume of 100 µL in the presence of the cells’ corresponding media. The cells were allowed to migrate toward the bottom chamber wells which were filled with 160 µL of 10% FBS growth medium, as a source of chemoattractant (directional migration). The cells were then placed in the RTCA DP instrument and invasion readings were taken every 5 minutes for 24 hrs.

The flow cytometry data was analyzed with a one-way ANOVA test using the Sidak’s correction for multiple comparisons to compare the expression of TK1 on the surface of the siRNA treated cells vs. isotype control. P-value significance was set at ≤0.05. Error bars represent the standard error of the mean. Statistical analyses were performed using Prism software.

To assess TK1 silencing on the surface of A549 cells, a cell line known to express high levels of membrane TK1, we stained the control and siRNA treated cells with antibodies against TK1. We chose to quantify silencing by flow cytometry because TK1 is a known cytoplasmic protein and we wanted to directly assess its expression on the cell membrane only. Flow cytometry revealed that A549 cells treated with siRNA targeting TK1 displayed decreased expression of TK1 on the surface. Cells treated with siRNA 58 and siRNA 59 showed significant reduction of TK1 on the surface, while siRNA 60 did not show a significant reduction (Figure 1). Cells treated with siRNA 58 had the most dramatic silencing in TK1 surface expression with a 93.82% reduction in surface expression compared to cells treated with control siRNA. Cells treated with siRNA 59 showed a 15.98% reduction at 60 hours post transfection.

To compare the invasion potential of A549 cells, with and without TK1 surface expression in vitro, we performed an invasion assay with A549 cells treated with control siRNA, siRNA 58, siRNA 59, and siRNA 60. In our study, the invasion potential of cells was measured by the cells crossing a layer of Matrigel from serum-free medium towards complete medium, as they seek nutrients. We performed the experiment in an xCELLigence RTCA DP instrument, which is used to determine real time invasion over a period of time. In our results, we observed that when TK1 is highly silenced on the membrane, the

invasion potential is reduced. In fact, only cells treated with siRNA 58, which had the most reduction of membrane TK1 expression, showed a significant reduction in invasion potential. Cells treated with siRNA 59 and siRNA 60 were statistically equally invasive to cells treated with control siRNA (Figure 2). We observed no change in cell viability before the invasion assay. Due to the short window of time post transfection, we did not evaluate growth rates. However, cell counts were similar between all transfected samples, suggesting no change in proliferation rates.