Cck 8 assay

The cell viability of HuCCT1, RBE, and
HCCC-9810 cells was determined using the CCK-8 assays (Vazyme) following
the manufacturer’s instructions. Briefly, HuCCT1, RBE, and
HCCC-9810 cells were seeded into 96-well plates at a density of 1
× 10⁴ cells/well, followed by exposure to a gradient
concentration of 5S and TMG for 48 h. To analyze the effect of H₂O₂ on cell proliferation, the cells were seeded
into 96-well plates with 2 × 10⁴ cells/well, followed
by exposure to a gradient concentration of H₂O₂ for 12 h. The cells seeded into 96-well plates with 5 × 10³ cells/well were subjected to 5 mM cell-permeable α-ketoglutarate
for the indicated time. Finally, 100 μL/well RPMI 1640 medium
containing 10% CCK-8 was replaced into the test well and incubated
at 37 °C for 1 h. Absorbance was then measured at a wavelength
of 450 nm.

For cell growth analysis, CRC cells (1000 cells/well) were resuspended in DMEM and cultured at 37 °C in a humidified CO₂ incubator for 10 days. Colonies were stained with 0.05% crystal violet, and those that were >1 mm in diameter were counted. The viability of cells was assessed by CCK8 assays (Vazyme Biotech), according to the manufacturer’s instructions.

The cell proliferation of the HTR-8/SVneo cell line was assessed via CCK-8 assays (Vazyme Biotech). After 48 h of transfection, the mixed liquor containing 90 μl of DMEM and 10 μl of CCK-8 solution was added to each well, into which 5000 cells were placed, followed by 2-h incubation. Absorbance (A) at 450 nm was then assessed via a SYNERGY H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc, Winooski, Vermont, USA).