Edu incorporation assay kit

To determine cell proliferation rates, we utilized the EdU incorporation assay kit (C0075L, Beyotime). Briefly, cells were treated with anlotinib-containing DMEM or RPMI-1640 medium in six-well plates. After treatment, cells were incubated with the EdU working solution for 2 h, fixed with 4% paraformaldehyde, and subsequently stained with Azide 555 as well as Hoechst 33,342. The percentage of fluorescence-positive cells was then calculated to assess the proliferation rate.

EdU incorporation assay kit (C0075L, Beyotime) was applied to determine cell proliferation rate. In short, DMEM or RPMI‐1640 medium containing apatinib and/or melittin was added to six‐well plate seeded with ATC cells. Cells were incubated with EdU working solution for 2 h after treatment, and then fixed with 4% paraformaldehyde. Subsequently, Azide 555 was added to the above cells treated with 0.5% Triton X100 and incubated for 30 min. Then cells were stained with Hoechst 33342. Finally, the percentage of fluorescence‐positive cells was calculated to assess the rate of proliferation.
Colony formation assay was employed as well. DMEM or RPMI‐1640 medium containing apatinib and/or melittin was added to six‐well plate seeded with low‐density ATC cells for 7 days. The colonies were stained with 0.1% crystal violet methanol solution for observation.