Fig. 4.
Increased VEGF autocrine is responsible for ATOH8 upregulation in a shear stress environment. a Heat map of aberrantly expressed cytokines and cytokine receptors in endothelial cells undergoing LSS (6 dyn/cm2, 24 h) from GSE52211. b, c Enzyme-linked immunosorbent assay (ELISA) measurement of human VEGF protein levels in LoVo and SW480 m-CTCs medium, treated with size gradient (0, 5, 10, 20 dyn/cm2; 30 min) (b) and time gradient (10 dyn/cm2; 0, 15, 30, 60 min) (c) LSS. d, e WB analysis of VEGF expression in LoVo and SW480 m-CTCs treated with size gradient (0, 5, 10, 20 dyn/cm2; 30 min) (d) and time gradient (10 dyn/cm2, 0, 15, 30; 60 min) (e) LSS. f Live/dead cell vitality assay for cell death rate in suspended LoVo and SW480 cells treated with or without LSS (10 dyn/cm2, 30 min) and VEGF (10 ng/mL). g WB analysis of expression level of ATOH8, HK2, BAX and BCL2 in suspended LoVo and SW480 cells treated with 10 ng/mL VEGF for 24 h. h Upper, representative immunofluorescence images of ATOH8 expression in suspended LoVo and SW480 cells treated with 10 ng/mL VEGF for 24 h. Down, quantification of fluorescence intensity. i Live/dead cell vitality assay in suspended LoVo and SW480 cells treated with LSS (10 dyn/cm2, 30 min), with or without 10 ng/mL VEGF and 5 μg/ mL bevacizumab. j WB analysis of expression level of ATOH8, HK2, BAX and BCL2 in suspended LoVo and SW480 cells treated with or without LSS (10 dyn/cm2, 30 min) and with or without 5 μg/ mL bevacizumab. k Suspended LoVo and SW480 cells transfected with ctrl or si-ATOH8 were seeded in low attachment 6-well plate and treated with 10 ng/mL VEGF for 24 h, and the expression of ATOH8, HK2, BAX and BCL2 were performed. l Live/dead cell vitality assay for cell death rate in suspended LoVo and SW480 cells transfected with ctrl or si-ATOH8 and then treated with or without VEGF (10 ng/mL). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001