Journal: Journal of Gastrointestinal Oncology

Article Title: A novel model based on protein post-translational modifications comprising the immune landscape and prediction of colorectal cancer prognosis

doi: 10.21037/jgo-24-45

Figure Lengend Snippet: Phosphorylated CMET suppresses the viability of CRC cells. (A) Immunoblot assessment of p-met protein levels. Plasmid DNA of CMETY1235E-HA, CMETY1235F-HA, or empty-plasmid respectively transfected into CRC cells. The samples were derived from the same experiment and blots were processed in parallel. (B-E) Growth curve (B); colony formation (C), scale bars: 2 mm; transwell experiments (D), scale bars: 100 µm; wound-healing assay (E), scale bars: 20 µm of CRC cells in the Control, CMETY1235E, CMETY1235F groups. Results are presented as mean ± SD for three independent experiments. *, P<0.05; **, P<0.01; and ***, P<0.001, compared with control. Observation methods: CRC cells were screened with 2 µg/mL purinomycin after transfected with plasmid 24 h. When their growth reached 80% confluence, we first collected cells and extracted the total cell protein using RIPA Lysis Buffer (#P0013B, Beyotime Biotechnology, Shanghai, China) to observe the phosphorylated state of the protein. Secondly, CRC cells were seeded into 96-well plates, 6-well plates, and 8-µm pore inserts for the optimal time, then we monitored their growth by methyl thiazolyl tetrazolium (Topscience Biological Technology, Shanghai, China). The cells were impregnated with 0.1% crystal violet staining solution (#G1062, solarbio, Beijing, China) to observe colony formation and migration. Meantime, CRC cells were seeded into the 2-well ibidi culture, inserted 48 h for wound healing analysis. CRC, colorectal cancer; SD, standard deviation.

Article Snippet: Cell viability assays were performed using the methyl thiazolyl tetrazolium (MTT) colorimetric assay (Topscience Biological Technology, Shanghai, China) at a wavelength of 490 nm by spectrophotometry.

Techniques: Western Blot, Plasmid Preparation, Transfection, Derivative Assay, Wound Healing Assay, Control, Lysis, Staining, Migration, Standard Deviation