Edu imaging kit

MGC 803 cells were plated at a density of 8.0 × 10⁴ cells/ml on a 96-well plate and allowed to adhere to plates overnight. EdU staining was carried out using the EdU imaging kit (RiboBio Co., China). Briefly, cells treated with galangin (20 μM) were first labeled with 50 μM EdU at 37°C for 2 h. Subsequently, they were fixed with 4% PFA for another 30 min, and incubated with 1 × PBS solution containing 0.5% Triton X-100 for 10 min. After washed with 1 × PBS solution, the cells were incubated with 100 μL dying solution for 30 min in the dark. Finally, the nuclei were stained with Hoechst 33342 solution for another 30 min. Fluorescent images were captured by fluorescence microscopy (IX81). Data were analyzed by using ImageJ software.

Cells were cultured on a 96-well plate and transfected with siRNAs or mimics for various times. Cell viability was then measured by the CCK-8 kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. For CCK8 assay, 1000 per well cells were seeded into 96-well plates 24–48 h after transfection and the absorbance were detected by microplate reader at 450 nm. Cell proliferation capacity was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. EdU is a thymidine analogue that can be inserted into the replicating DNA instead of thymidine (T) during cell proliferation. The fluorescent dye Apollo® binds specifically to EdU which has inserted into DNA and could be detected by excitation light at 488 nm. Green light represents the growing cell. Hoechst stained cell nuclei represent the total number of cells. In EdU incorporation assay, 1 × 10⁴–1.5 × 10⁴ cells were seeded into 96-well plates 24 or 48hplates 24 or 48 h after transfection and the next steps were following the manufacturer’s protocol of EdU Imaging Kit (Ribobio, China), the fluorescent images were acquired by the fluorescence microscope (Olympus, Japan). All the experiments were performed for at least three times.

EdU assay was used to detect the DNA synthesis of growing PTC cells by using the EdU imaging kit (RiboBio, China), and the EdU assay steps were performed following the manufacturer’s instructions.