Dakocytomation

HeLa cells (10 × 10⁵) were seeded in a 60 mm Petri dish and were allowed to grow for 24 h. FSK of FSK-20 μM, FSK-40 μM, FSK-80 μM and FSK-100 μM concentrations were added to the culture medium and the cells were incubated for an additional 24 h. Thereafter, the cells were harvested with trypsin/ Ethylenediaminetetraacetic acid (EDTA), fixed with ice-cold 70% ethyl alcohol (EtOH) at −80 °C for overnight. On the next day the fixed cells were washed with PBS and incubated with 1 mg/mL RNase A solution (Sigma) at 37 °C for 30 min. Subsequently, the cells were collected by centrifugation at 2000 rpm (Heraeus Sorvall swinging bucket rotor (model #75002000), max speed: 4700 rpm, Heraeus Multifuge 1S-R, Thermo Scientific) for 5 min and further stained with 250 µL DNA staining solution (10 mg propidium iodide (PI), 0.1 mg trisodium citrate, and 0.03 mL Triton X-100 dissolved in 100 mL sterile Milli-Q water at room temperature for 30 min in the dark). The DNA content of 20,000 events was measured by flow cytometry (DAKOCYTOMATION, Beckman Coulter, CA, USA). The resultant histograms were analysed using Summit software.

5X 10⁵ HNGC2 and U87 cells were seeded in 60 mm dish and allowed to grow for 24 h followed by transfection with miR-203 plasmid construct. Cells were harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 40^◦C for 30 min, washed with PBS and incubated with 1mg/ml RNase A solution (Sigma) at 37°C for 30 min. Further cells were collected by centrifugation at 2000 rpm for 5 min and stained with 250 μL of PI solution (Propidium Iodide) (10 mg of PI, 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 at room temperature for 30 min in dark. The DNA contents of 10,000 events were measured by flow cytometer (Dakocytomation, Beckman Coulter). Histograms were analyzed using summit software.