Hoechst 33342 staining assay

The potential toxicity of RUT at the nuclear level was tested by applying the Hoechst 33342 staining assay protocol according to the manufacturer’s (Thermo Fisher Scientific, Inc., Waltham, MA, USA) recommendations. In brief, the RPMI-7951 and SK-MEL-28 cells were seeded in 12-well plates (10⁵ cells/1.5 mL/well) and treated with three selected concentrations (1, 10, and 50 µM) of RUT in DMSO for 24 h. After the stimulation period, the media was removed, and the staining solution diluted at 1:2000 in PBS was added (500 µL/well). The plates were incubated for 10 min at room temperature, protected from light. Finally, the staining solution was washed with PBS and the pictures were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA) and analyzed by the means of Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, USA). Staurosporine (STP) 5 µM was selected as a positive control for apoptosis. The apoptotic index (AI) was calculated according to a formula described by Xu et al. [24 (link)]: AI=Number of apoptotic cellsTotal number of cells × 100

The potential toxicity of the samples (5FU, PBT, and PBT-5FU) at the nuclear level was evaluated by using the Hoechst 33342 staining assay protocol according to the manufacturer’s (Thermo Fisher Scientific, Inc., Waltham, MA, USA) recommendations and to our previous research [14 (link)]. In brief, the malignant cells were seeded in 12-well plates (100,000 cells/1.5 mL/well) and treated with test samples in solvent (media) for 24 h. After the stimulation period, the media was removed, and the staining solution diluted at 1:2000 in PBS was added (500 µL/well). The plates were incubated for 10 min at room temperature, protected from light. Finally, the staining solution was washed with PBS, and the pictures were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA) and analyzed by the means of the Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, USA). Staurosporine (STP) 5 µM was selected as a positive control for apoptosis.