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Total 60,000 cells were cultured in 1 mL medium for duration of 18 hours, followed by transfection. After duration of 24 hours, 50 nmol/L MTX was added into cells. Apoptosis was measured after 6 hours of MTX treatment. Under dark conditions, 5 μg/mL Annexin V‐fluorescein isothiocyanate (FITC) and 5 μg/mL propidium iodide (PI) (Sigma‐Aldrich, Madrid, Spain) were applied to stain cells for 15 minutes. A flow cytometer (Becton‐Dickinson, Franklin Lakes, NJ, USA) was adopted with the aim to examine cell apoptosis.
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