Cho k1

Human ovarian cancer cells (SKOV-3; described as “cancer cells”) and noncancer Chinese hamster ovary cells (CHO-K1; described as “normal cells”) were obtained from the American Type Culture Collection (ATCC, London, UK). Cells were cultured as a monolayer in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, USA; for SKOV-3 cells) and Ham’s F-10 Nutrient Mix (F-10, Sigma-Aldrich, USA; for CHO-K1 cells) containing 2 mM L-glutamine, 10% fetal bovine serum (FBS, Sigma-Aldrich), 20 U penicillin, and 20 µg streptomycin/ml (Sigma-Aldrich) at 37°C in a humidified incubator with 5% CO₂. For the experiments, the cells were washed with Dulbecco’s phosphate-buffered saline (PBS, Bioshop, UK) and detached from the flask’s surface using 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, Poland).

Chinese Hamster Ovary (CHO-K1, Sigma) cells were cultured in Ham's F-12 Nutrient Mix supplemented with 1 mM l-glutamine, 100 U ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin, 1 mM sodium pyruvate, and 10% v/v heat-inactivated Fetal Bovine Serum (FBS, Gibco ThermoFisher Scientific). CHO cells stably transfected with the PAR biosensors cloned into pcDNA3.1(+) were cultured in complete F-12 medium with 600 µg ml⁻¹ geneticin selective antibiotic (G418 Sulfate, Gibco ThermoFisher Scientific). The cells were grown in a T75 cell culture flask (Nunc) in a humidified cell culture incubator with 5% CO₂ at 37 °C. Cells at ~ 80–90% confluency were detached with phosphate-buffered saline (PBS, Gibco ThermoFisher Scientific) solution supplemented with 1 mM EDTA (Fisher Scientific), centrifuged at 180 × g for 5 min, and sub-cultured as appropriate.
Human embryonic kidney (HEK)-293 cells (ATCC) stably transfected with PAR4-eYFP-pcDNA3.1(+)^{52 (link)} were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco ThermoFisher Scientific) supplemented with 1 mM sodium pyruvate, 100 U ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin, 2 mM l-glutamine, 10% v/v FBS, and 600 µg ml⁻¹ G418. Cells at ~ 80–90% confluency were dissociated with trypsin–EDTA (0.25%, Gibco ThermoFisher Scientific), centrifuged at 180 × g for 5 min, and sub-cultured as appropriate.

Standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma Aldrich, Prague, Czech Republic) was used according to the manufacturer´s protocol on the CHO-K1 (Chinese hamster ovary, ECACC, Salisbury, UK) in order to compare the effect of different compounds within the series. The cells were cultured according to ECACC recommended conditions and seeded in a density of 8000 per well as described previously [52 (link)]. Briefly, tested compounds were dissolved in DMSO (Sigma Aldrich, Prague, Czech Republic) and subsequently in the growth medium (F–12) so that the final concentration of DMSO did not exceed 1% (v/v). Cells were exposed to a tested compound for 24 h. Then the medium was replaced by a medium containing 10 μM of MTT and cells were allowed to produce formazan for another approximately 3 h under surveillance. Thereafter, medium with MTT was sucked out and crystals of formazan were dissolved in DMSO (100 µL). Cell viability was assessed spectrophotometrically by the amount of formazan produced. Absorbance was measured at 570 nm with 650 nm reference wavelength on Synergy HT (BioTek, Winooski, VT, USA). IC₅₀ was then calculated from the control-subtracted triplicates using non-linear regression (four parameters) of GraphPad Prism 9 software. Final IC₅₀ and SEM values were obtained as a mean of three independent measurements.