Modfit lt software 5

The inhibitory concentrations of the cell cycle inhibitors were adjusted using cell cycle analysis and apoptosis experiments via flow cytometry. Experiments were performed at the FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) cytometer with facsdiva software 7.0 (BD Biosciences).
For S phase analysis, 5 × 10⁵ cells in suspension were washed and fixated with 500 μL of 70% methanol (Carl Roth, Karlsruhe, Germany) overnight at 4 °C. RNA digestion was conducted with 25 μg Ribonuclease A (Sigma‐Aldrich) for 20 min at 37 °C. DAPI staining was as performed with 250 ng DAPI (Sigma‐Aldrich) for at least 30 min at room temperature (RT). Signal intensity was measured with the cytometer FACS Canto II (BD Biosciences), facsdiva software 7.0 (BD Biosciences) and analyzed with ModFit LT software 5.0 (Verity Software House, Topsham, ME, USA).
Cell viability was analyzed via flow cytometry. All incubation steps were performed at 4 °C or on ice. Cell suspensions with 5 × 10⁵ cells were washed with PBS (Sigma‐Aldrich). Annexin V‐FITC (Immunotools GmbH, Friesoythe, Germany) staining was performed according to the manufacturer's protocol. Prior to the measurement, 50 ng DAPI (Sigma‐Aldrich) was added to the cells and incubated for 1 min. Dot plots were created with Flowing Software 2.5.1 (Turku Centre for Biotechnol., Uni. Turku, Finland).

The cell cycle phase arrest was determined by flow cytometry according to the protocol provided by the manufacturer, using the BD cell cycle kit (Pharmingent, London, UK). Briefly, PC-3 cells were harvested at 1 × 10⁶ cells/mL and the cell suspension was transferred into a 15 mL falcon tube and centrifuged at room temperature at 1500× g for five minutes.
The supernatant was discarded carefully, and 1 mL of buffer solution was added. The cells were vortexed at low speed and the number was adjusted to 1 × 10⁶ cells/mL by using a buffer solution. Subsequently, trypsin buffer was added to the cells and incubated for 10 min in the dark. Two hundred microliters of solution B (trypsin inhibitor and RNase buffer) was added into the mixture. The cells were tapped slowly to allow mixing and left at room temperature for 10 min. Finally, the cells were then stained with 200 µL of cold solution C (propidium iodide stain solution). The solution was mixed and left at room temperature for 10 min in the dark. The cells were strained using a cell strainer, transferred into a 17 × 100-mm tube and gently tapped. The cells were immediately analyzed using the flow cytometer (BD Accuri, New Jersey, NJ, USA) by analyzing at least 10,000 cells per sample. The percentage of cells in the G1, S, and G2 phases was analyzed by the ModFit LT software 5.0 (Verity Software House, Topshom, ME, USA).