Tl4 photomicroscope

CCK-8 (Cell Counting Kit-8) assay was carried out as follows: 5,000 cells were seeded in each well of the 96-well plates and cultured overnight at 37°C. After treating with myricetin for 24 h, the cells were incubated with CCK-8 reagents (Biosharp, Beijing, China) for 1 h at 37°C. The light absorbance at 450 nm was measured by a microplate reader (SpectraMax M2, Molecular Devices, Shanghai, China).
The EdU (5-Ethynyl-2′-Deoxyuridine) proliferation assay was determined by using EdU detection kits (#C10310-1, Ribobio, Guangzhou, China) according to the manufacturer’s instruction. 5,000 cells were seeded in 96-well plates per well and cultured overnight. The cells were then treated with myricetin for 24 h. Then, the cells were stained with 50 μM EdU medium for 2 h, immobilized with 4% polyoxymethylene for 30 min, treated with 2 mg/ml glycine for 5 min, and permeated by 0.5% TritonX-100 for 10 min. After that, the cells were mixed with Apollo 567 for 30 min at room temperature in the dark and permeated again by 0.5% TritonX-100 for 20 min. Last, cells were maintained in Hoechst 33,342 for 30 min at room temperature in the dark. Cells were detected by the fluorescence microscope (Olympus TL4 photomicroscope, Japan). Proliferative cell nuclei were stained red fluorescence, and all cell nuclei showed blue fluorescence. The cell proliferation rate was analyzed by ImageJ.

The cells were grown in a 6-well plate until they reached 90% confluence. Scratches were made in the cell monolayer using a 10 μL sterile pipette tip. Next, the cells were washed with PBS, and the medium was replaced with fresh medium supplemented with 1% FBS to inhibit cell proliferation. The scratched areas were photographed at the indicated time points using an Olympus TL4 photomicroscope (Olympus, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell proliferation was evaluated by EdU assay using a commercially available kit (#C10310-1, Ribobio, Guangzhou, China). Cells were seeded in 96-well plates at 5,000 cells per well, and incubated for 24 h at 37°C before treating with SIN. After 24 h of drug treatment, cells were incubated with 50 μM EdU medium for 2 h. Cells were then immobilized with 4% polyoxymethylene for 30 min, treated with 2 mg/ml glycine for 5 min, and permeated by 0.5% TritonX-100 for 10 min. Cells were incubated in Apollo 567 for 30 min at room temperature in the dark and permeated by 0.5% TritonX-100 for 20 min. At last, cells were incubated with Hoechst 33,342 for 30 min at room temperature in the dark. PBS was used for washing between each step. Images were collected by a fluorescence microscope (Olympus TL4 photomicroscope, Japan).