Fig 2: EHD1 induces VEGFA expression and angiogenesis in a ß2AR-dependent manner in NSCLC. a Microarray analysis showed an obvious overlap between ß2AR-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 ß2AR expression in A549 and NCI-H1650 cells after EHD1 knockdown. d Immunoblotting analysis of ß2AR 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

Fig 3: EHD1 expression is associated with the response to erlotinib treatment and stemness in lung cancer.a IHC staining yielded scores ranging from 0 to 3, which are representative of the amount of EHD1 and CD133 in lung cancer specimens. A score of 0–1 represented low expression, and 2–3 represented high expression. b The percentages of patients with high expression (black bar) and low expression of EHD1 (grey bar) were assigned according to different responses to EGFR-TKI (responder, n = 12; nonresponder, n = 11). Numbers near bars represent the percentage of patients for each condition. *P < 0.05. c Mean OS of patients with high levels of EHD1 (n = 11, OS of 26 mo) and low levels of EHD1 (n = 12, OS of 39 mo) taking EGFR-TKI treatment. Mean PFS of patients with high levels of EHD1 (n = 11, PFS of 11 mo) and low levels of EHD1 (n = 12, PFS of 30 mo) taking EGFR-TKI treatment. P = 0.049. d Patients with high CD133 expression were accompanied by increased expression of EHD1 protein. The expression levels of EHD1 were classified into two groups according to the IHC scoring: low-expression group (score 0 and 1) and high-expression group (score 2 and 3). e EHD1 expression in human lung cancer cell lines. f The correlation between EHD1 expression and IC50 of erlotinib in human lung cancer cell lines

Fig 4: The interaction between EHD1 and Wnt4. (a) The protein levels of Wnt4 and β-catenin with Ad-LacZ or Ad-Flag-EHD1 at the MOI indicated and after treatment with 8-Br-cAMP and MPA for three days were assessed by Western blot. *p < 0.05, **p < 0.01 compared with the control group or decidua group (n = 3, one-way analysis). (b) Images of the colocalization of EHD1 (red) and Wnt4 (green) in the control and RIF groups. Each of the two proteins was immunoprecipitated with anti-EHD1 antibody or anti-Wnt4 antibody from HEK293T cells (c) and HESCs (d), and the immunoprecipitates were subjected to Western blot analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 5: EHD1 impairs HESC decidualization in vitro by repressing the Wnt4/ß-catenin pathway. (a) 2D scatter plot of the principal component analysis (PCA) results. Samples in the control group (control), decidual (decidua) group and overexpression EHD1 group (EHD1) (n = 3) are represented by blue, orange and red dots, respectively. (b) Venn diagrams showing the overlapping genes and differentially expressed genes (DEGs) among the control, decidual, and EHD1 groups. (c) Hierarchical clustering of DEGs between different groups, the left panel with the control group vs the decidua group and the right panel with the decidua group vs the EHD1 group. (d, e) Gene Ontology (GO) functional classification of the DEGs and the KEGG pathway analysis between the decidual group and EHD1-overexpressing group. (f) FRKM expression by RNA-seq among the control, decidua, EHD1 groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)