Cell viability, scratch assay and reactive oxygen species
measurement
After the treatment with different concentrations of GS for 48 h, cells
were incubated with Cell Counting Kit 8 (CCK8) solution (10 μl/well,
Glpbio, Montclair, CA, USA) at 37°C for 40 min. The absorbance at 450 nm
for different wells was detected on a microplate reader
(Infinite
M200pro, TECAN, Zurich, Switzerland) to analyze cell viability,
according to our previous study (Li et
al., 2018). For the scratch assay, a straight scratch was made using a
P200 pipet tip in cells treated with GS for 48 h. After washing with
phosphate-buffered saline, the images of scratch area at 0 and 24 or 72
h were captured using the Olympus Viva View FL microscope (Tokyo, Japan)
and analyzed to calculate the rate of scratch closure for four fields
per dish from three independent experiments using ImageJ software
(Graham et al., 2018). Intracellular and
mitochondrial reactive oxygen species (ROS) generation of cells treated
with various concentrations of GS for 48 h were detected by
carboxy-H2DCFDA fluorescent probes (Beyotime Biotechnology) or MitoSOX
mitochondrial superoxide indicator (Thermo Fisher Scientific) using the
FACS-Calibur™ flow cytometer (BD Biosciences, San Jose, CA, USA).