Cell titer aqueous one solution

Replicon experiments were conducted as previously described [72 (link)]. Briefly, Huh7 cells were detached by trypsin, washed twice in ice-cold DEPC-treated PBS and re-suspended at 1 x 10⁷ cells / mL in DEPC-treated PBS. Subsequently 400 μL of cells was mixed with 2 μg of RNA transcript, transferred to a 4 mm gap electroporation cuvette (SLS, UK) and pulsed at 260 V, 25 ms pulse length in a Bio-Rad Gene Pulser (Bio-Rad, USA). Electroporated cells were recovered into 4 mL media, seeded into replicate 6-well tissue culture vessels, and replication measured at 24 h intervals using Nano-Glo luciferase assay system (Promega, USA). For inhibitor treatment the electroporated cells were seeded into replicate 24-well plates, allowed to adhere for 24 h before the media was replaced with fresh media containing antipain, AEBSF, leupeptin, (Sigma-Aldrich, USA), warfarin, dabigatran etexilate (Merck Life Sciences, USA) or apixiban (Insight Biotechnology, UK), at the indicated concentrations. The cell viability experiments were conducted by seeding cells into 96-well plates, allowing to adhere for 24 h before addition of a serial dilution of protease inhibitors and measurement of cell viability 72 h later using the CellTiter AQueous One solution (Promega, USA), following manufacturer’s instructions.

To assess proliferation, we performed MTS colorimetric assays in KLHL14-depleted and control-transfected NCI-H2052 and NCI-H2452 cells, stimulated or not with TGF-β. Briefly, 2 × 10³ cells/well were plated into 96-well microplates and starved for 24 h prior to TGF-β exposure. CellTiter® AQueous One Solution (Promega Corporation Madison, WI, USA) was used following supplier's guidelines. Cell growth was determined by the amount of formazan produced, analyzed by measuring the absorbance at 492 nm in a PerkinElmer VICTOR X5 2030 Multilabel plate reader (PerkinElmer, Waltham, MA, USA). The analysis was carried out at least in triplicates and the experiments performed 3 times.
For colony formation assay, 1 × 10³ NTC or KLHL14-depleted cells/well were seeded onto 6-well plates in complete media, allowed to attach and incubated until colonies were formed. Approximately 6–8 days after, colonies were subsequently washed with PBS, fixed with absolute methanol, and stained at room temperature for 30 min with crystal violet solution prepared at 0.5% in 25% methanol and 75% distilled water. Colonies were then additionally washed with PBS, dry and counted manually for statistical analysis.

Tumor cells were plated in 96-well plate (Falcon 35–3075) in 180 μL growth media at 3000–5,000 cells/well. The cells were incubated overnight at 37 °C with 5 % CO₂. On Day 2, dilutions of testing articles were added to the cell plate (20 μL/well) in triplicate wells. Untreated control wells (for 0 % control) and wells treated with 10 μM staurosporin (for 100 % kill) were included in each plate. The plates were incubated for 3–5 days at 37 °C with 5 % CO_2. To quantify live cells, media was removed and 1x Cell Titer Aqueous One Solution (Promega, G3581) diluted in Opti-mem media (Invitrogen # 31985–070) was added to plates and the plates were then incubated for one hour at 37 °C. The OD at 490 nm was read on a M5 Spectramax plate reader (Molecular Probes). Percent inhibition was calculated based on 100 % kill and untreated control wells using the following formula: 100x (0 % control - treated)/(0 % control - 100 % kill). For cells grown in suspension such as SNU5, cells were plated in 96-well plate in 180 μL medium at 10,000 cells/well and incubated overnight at 37 °C with 5 % CO₂. The same protocol as above was used for treatment and data processing except live cells were detected by adding forty μL of Cell Titer Aqueous One Solution to each well and the plates were then incubated for one hour at 37 °C. The experiments were repeated at least twice for each cell line.