Fig. 3

EHD1 induces VEGFA expression and angiogenesis in a β2AR-dependent manner in NSCLC. a Microarray analysis showed an obvious overlap between β2AR-dependent gene expression and EHD1-regulated gene expression. The pseudocolor represents the intensity scale for the EHD1 ShRNA vector versus the control, as calculated by log2 transformation. b Western blot of EHD1 expression in human lung cancer cells. c Western blot analysis of β2AR expression in A549 and NCI-H1650 cells after EHD1 knockdown. d Immunoblotting analysis of β2AR expression in A549 and NCI-H1650 cells after EHD1 reexpression. e A549 cells were transfected with vector or ShEHD1 and then treated with DMSO or ISO, and the EHD1 and VEGFA protein levels were assessed by immunoblotting. f A549 cells were incubated overnight with serum-free 1640 medium and corresponding reagents, and their CMs were used to test the level of VEGFA secretion by ELISA. g The viability of HUVECs was detected by CCK8 assay. HUVECs in 96-well plates were incubated with CMs from A549 cells. h CMs were added to the lower chamber, and HUVECs were seeded on the upper chamber. After 24 h of incubation, HUVEC migration was assessed by counting the cells on the lower surface of the membrane; from left to right, the lanes show Ctrl, Sh and Sh/ISO. Scale bar, 100 μm. i HUVECs were incubated with CMs from A549 and NCI-H1650 cells in 48-well plates, and their tube formation abilities were evaluated based on the number of tubes per field