Propidium

A total of 2 × 10⁵ cells/well were seeded in 6-well plates. The cells were digested using trypsin for 3 min before the samples were centrifuged to collect the cells. After washing it 3 times with PBS, the sample was resuspended in 70% ethanol. Next, the cells were fixed at 4 °C for 12 h in the 70% ethanol, washed with PBS again, centrifuged at 1000 rpm for 5 min, and stained with propidium for 30 min in a dark environment (Beyotime, Shanghai, China). Then, the cell cycle was detected using a flow cytometer.

Following transfection, CAL-27 and SAS cells were trypsinized using a trypsin solution without ethylenediaminetetraacetic acid. The cells were then collected by centrifugation at 1000× g for 5 min, washed with ice-cold PBS, and fixed with 70% cold ethanol for storage at 4 °C for 24 h. After another round of centrifugation and washing with cold PBS, the cells were treated with 25 μL Propidium and 10 μL RNase A (Beyotime, Shanghai, China) at 37 °C in the dark for 30 min. For CD133 detection, cells were obtained and stained with anti-CD133 (17–1338-42, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C in the dark for 30 min. Cell-cycle analysis was conducted using NovoExpress [Version 1.6.2, Agilent, Santa Clara, CA, USA], and the CD133 was analyzed using CytExpert software [Version 2.4.0.28, Beckman Coulter, Brea, CA, USA].