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).