Cell proliferation was measured by Cell Counting Kit-8 (CCK8) assays and EdU assays.
For CCK8 assays, cells were seeded in 96-well plates at a density of 2000 cells/well and then were incubated for 12 h to allow cell attachment. Then, 10 μL of CCK-8 solution (Bimake, Cat# B34304) was added to each well on days 1, 2, 3, 4, 5 and 6, which was followed by another 2 h incubation. The optical density was then measured at 450 nm using a microplate reader.
For EdU assays, cells were plated at a density of 20000/dish in confocal dishes. After 24 h of incubation, cells were treated with EdU reagent (RiboBio, Cat# C10310-1) for 2 h according to the manufacturer's instructions and then were xed with 4% paraformaldehyde. One hundred microliters of 1X Apollo®567 staining reaction solution and Hoechst 33342 (RiboBio, Cat# C10310-1) was added to each dish and then was incubated for 30 min at room temperature on a decolorization shaker. Cells were then visualized using a BX63 automatic intelligent uorescence microscope (Olympus, Tokyo, Japan).
For CCK8 assays, cells were seeded in 96-well plates at a density of 2000 cells/well and then were incubated for 12 h to allow cell attachment. Then, 10 μL of CCK-8 solution (Bimake, Cat# B34304) was added to each well on days 1, 2, 3, 4, 5 and 6, which was followed by another 2 h incubation. The optical density was then measured at 450 nm using a microplate reader.
For EdU assays, cells were plated at a density of 20000/dish in confocal dishes. After 24 h of incubation, cells were treated with EdU reagent (RiboBio, Cat# C10310-1) for 2 h according to the manufacturer's instructions and then were xed with 4% paraformaldehyde. One hundred microliters of 1X Apollo®567 staining reaction solution and Hoechst 33342 (RiboBio, Cat# C10310-1) was added to each dish and then was incubated for 30 min at room temperature on a decolorization shaker. Cells were then visualized using a BX63 automatic intelligent uorescence microscope (Olympus, Tokyo, Japan).