Cell proliferation assay
The MTT assay was used to assess the cell growth rate. Briefly, cells
were plated in 96-well plates (4x103 per well) and
subjected to different treatments. The detailed procedures have been
previously described by Dou et al [45]. For colony formation assay,
cells were cultured in 24-well plates (800 cells/ well) and treated with
different treatments. After 2 weeks, the colonies were fixed with 4%
paraformaldehyde and stained with Giemsa. The visible colonies were
photographed by Molecular Imager Gel Do XR + System (Bio-Rad, Hercules,
CA, USA) and counted using ImageJ software (National Institutes of
Health, Bethesda, MD, USA). The detailed procedures have been previously
described by Wang et al [38]. The 5-ethynyl-20-deoxyuridine (EdU)
labeling assay was performed in 24-well plates (2 x
104 cells) using the EdU cell Proliferation Assay Kit
(Ribobio, Guangzhou, China). After different treatments, 10 μmol/L EdU
was added to the cells, and the cells were incubated for 12 hours at
370C. Cells were fixed with 4% paraformaldehyde. DAPI
was then added for nuclear staining followed by imaging with a
fluorescence microscope.