Fig 2: Inhibition of MST1/2 rescues the effect of MT2A on CRC cells. A XUM-MP-1 is a specific inhibitor of MST1/2. 2 µM XUM-MP-1 was added and cultured for 6 h. The results showed that both p-MST1 and p-YAP1 were significantly inhibited in HCT8 and HCT116 cells. B, C Colony formation assays showed that XUM-MP-1 promoted colony formation in HCT8 and HCT116 cells as well as rescued the inhibitory effect of MT2A overexpression. D The CCK-8 assay showed that XUM-MP-1 significantly increased the cell proliferation rate of MT2A-overexpressing cells. E IHC staining of MT2A showed that the MT2A overexpression group consistently overexpressed MT2A in subcutaneous tumors. P-MST1 and p-YAP1 were upregulated in the MT2A overexpression group compared to the control group. F Consistent with subcutaneous tumors, IHC staining of liver metastasis tumors showed that MT2A overexpression increased the expression of MT2A, p-MST1 and p-YAP1. Scale bar = 100 µm, OE, overexpression. *p < 0.05, **p < 0.01

Fig 3: MT2A promotes Hippo signaling in CRC cells. A RNA sequencing was used to evaluate the gene expression level between MT2A-overexpressing and control HCT8 and HCT116 cells. B With a cutoff of p < 0.05 and |log2fold change|> 2, 106 genes were selected after MT2A overexpression in both cell lines, including 93 upregulated genes and 13 downregulated genes. C GSEA showed that MT2A overexpression significantly enriched Hippo signaling in HCT8 and HCT116 cells (normal p value < 0.05, NES > 1.0). D We used a Proteome Profiler Array Human Phospho-Kinase Array to identify the key variations in phosphokinases after transfection of MT2A. This array is a rapid and sensitive product used to detect relative levels of phosphorylation of 43 kinase phosphorylation sites and GAPDH protein. The A-B5 and B6 sites represent the expression levels of p-MST1 and p-MST2, respectively. MT2A overexpression promoted the phosphorylation of MST1 and MST2. E In HCT8 and HCT116 cells, the expression levels of p-MST1, MST1, p-LATS2, LATS2, p-YAP1, and YAP1 were determined by Western blot analysis. MT2A overexpression increased the phosphorylation of p-MST1, p-LATS2 and p-YAP1 in both cell lines. F Immunofluorescence staining of YAP1 showed that MT2A overexpression inhibited nucleus location of YAP1 in HCT8 cells. OE overexpression. Scale bar = 5 µm