Edu labeling medium

96-well plates were seeded at 3000 cells per well and exposed to different treatments. 50 μM EdU labeling medium (RiboBio) was added and incubated for 2 h at 37 °C. The cells were then fixed with 4% cold methanol for 10 min. After being washed with PBS, cells were stained with anti-EdU working solution for 30 min and stained with Hoechst33342 for 10 min. Cells were observed using fluorescent microscopy. The percentage of EdU-positive cells was calculated from five random fields in three wells.

For CCK-8 assay, we seeded TPC-1 and K-1 cells after transfection in 96-well plates (1000 cells/well) and cultured them at 37 °C with five percent CO₂. The cell viability was examined every 24 h, in brief, 10 μL CCK8 solution was added into per well. After incubation for 2 h at 37 °C, the microplate reader was utilized to detect the absorbance of all wells at an optical density of 450 nm. We planted 500 cells in 6-well plates and cultured them at 37 °C with 5% CO₂ for 14 days in colony formation assay. Following being washed, the colonies were fixed in four percent paraformaldehyde for fifteen minutes, dyed by 0.1% crystal violet solution for 20 min, and finally counted manually. For Ethynyldeoxyuridine (EdU) assay, we seeded the transfected cells (density: 5000 cells/well) on ninety-six-well plates. Then, 50 μM EdU labeling medium (RiboBio, Guangzhou, China) was added to these wells and then incubated for 2 h under 5% CO₂ at 37 °C. After treated with four percent paraformaldehyde and 0.5% Triton X-100, cells were stained by Apollo Dye Solution. Then Hoechst 33342 was used to stain the nucleic acids within the cells. Ultimately, we used fluorescence microscopy (Nikon, Japan) to analyze and calculate the rate of EdU-positive cells.