Fig. 5
VEGFR2-AKT pathway contributes to VEGF induced ATOH8 expression in CRC m-CTCs. a The qPCR analysis of VEGFR2 expression in suspended LoVo and SW480 cells treated with size gradient (0, 5, 10, 20 dyn/cm2, 30 min, Left) and time gradient (10 dyn/cm2, 0, 15, 30, 60 min, Right) LSS. b Suspended LoVo and SW480 cells was treated with VEGFR2 inhibitors ZM323881 (0, 5, 10, 20 μM) or Apatinib (0, 5, 10, 20 μM), and the relative change in ATOH8 expression was analysed by WB. c, d Suspended LoVo and SW480 cells treated with or without 10 ng/mL VEGF and with or without 10 μM VEGFR2 inhibitor (ZM323881). The expression of ATOH8, HK2, BAX and BCL2 were analysed (c), additionally, cell death was tested by Live/dead cell vitality assay (10 dyn/cm2, 30 min) (d). e, f Suspended LoVo and SW480 cells with ATOH8 overexpression were treated with or without 10 μM VEGFR2 inhibitor (ZM323881). The expression of ATOH8, HK2, BAX and BCL2 (e) and the cell death (f) were examined by WB and live/dead cell vitality assay (10 dyn/cm2, 30 min) separately. g The ssGSEA of AKT or ERK signalling pathways in ATOH8high and ATOH8low group in the colorectal cancer metastasis cohort from GSE131418. h, i Suspended LoVo and SW480 cells treated with or without 10 ng/mL VEGF and with or without 10 μM AKT inhibitor (AZD5363). The protein levels of ATOH8, HK2, BAX and BCL2 were analysed by WB (h), additionally, cell death was tested by Live/dead cell vitality assay (10 dyn/cm2, 30 min) (i). j WB analysis of expression level of ATOH8, HK2, BAX and BCL2 detected in suspended LoVo and SW480 cells with ATOH8 overexpression, with or without 10 μM AKT inhibitor (AZD5363) for 24 h. k Live/dead cell vitality assay in suspended LoVo and SW480 cells after ATOH8 overexpression treated with LSS (10 dyn/cm2, 30 min), with or without 10 μM AKT inhibitor (AZD5363). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001