Fig. 3
ATOH8 induced intravascular survival via HK2-mediated glycolysis. a Single sample gene set enrichment analysis (ssGSEA) of gene-containing signature in ATOH8high and ATOH8low group in the colorectal cancer metastasis cohort from GSE131418 and the results of anoikis-related and key metabolic pathways were presented. b, c Overexpression of ATOH8 promoted lactate production (b) and HK2 enzyme activity (c) in suspended LoVo and SW480 cells, while opposite effects were observed when silencing ATOH8. d WB analysis of the expression level of glycolytic enzymes HK2, LDHA, and GLUT1 and apoptotic markers BAX, BCL2 in suspended LoVo and SW480 cells after overexpressing or silencing ATOH8. e qPCR analysis of HK2, LDHA, and GLUT1 expression in suspended LoVo and SW480 cells treated with LSS (10 dyn/cm2, 30 min). f Live/dead cell vitality assay for cell death rate in LoVo and SW480 mimic circulating tumour cells (m-CTCs) after overexpressing ATOH8 and treating with or without 1 mM 2-Deoxy-D-glucose (2-DG) or 2 nM 3-bromopyruvate (3-BrPA) (10 dyn/cm2, 30 min). g LoVo and SW480 cells were transfected with flag tagged ATOH8 and harvested for a chromatin immunoprecipitation (ChIP) assay to detect the enrichment of ATOH8 around the HK2 promoter. PCR products amplified with the indicated primers using anti-Flag antibody immunoprecipitated DNA (IP) as a template and anti-IgG or anti-histone H3 antibody immunoprecipitated DNA as negative or positive control. h Quantity of ChIP DNA pulled down. i The promoter of HK2 contains ATOH8 binding domains, and the binding sites of HK2 promoter wild-type or mutation vector were displayed. j Luciferase activity in 293 T cells when ATOH8 wild-type vector was co-transfected with HK2 promoter wild-type or mutation vector. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001