Fig 2: Cis EphB1 signaling mediates cancer cell dormancy after chemotherapy.A Western blot analysis of p-p38 in cisplatin-treated A549 cells at different time points; B Western blot analysis of proteins obtained from EphB1 transfected cells. A549 cells were transfected with EphB1 with or without treatment of p-p38 inhibitor SB203580. C Transwell migration and invasion assays. Representative microscopic images of cells that migrated through or invated through the transwell were shown. Cells with transfected with EphB1 with or without treatment of p-p38 inhibitor SB203580. The histogram graph was shown on the right. Data are presented as the mean ± S.D. from at least three separate experiments. Student’s t test, *P value < 0.05, **P value < 0.01; Scale bar = 100 µm; D Cell cycle analysis; E Flow cytometry analysis of apoptotic cells after treatment of cisplatin combined with p-p38 inhibitor. Representative flow cytometry plots using Annexin V-FITC/PI staining for apoptosis. The percentage of apoptotic cells were shown on the right. Data are presented as the mean ± S.D. from at least three separate experiments. Student’s t test, *P value < 0.05, **P value < 0.01.

Fig 3: Ligand-independent EphB1 mediates TGF-ß-activated N-cadherin. (A) Expression of mesenchymal molecules in lung cancer cells after transfection of EphB1 or knockdown of EphB1 quantified by real-time PCR. *p<0.05; **p<0.01; ***p<0.001. (B) Expression of CDH2 upon transfection of EphB1 measured by Western blot. (C) Expression of EphB1 upon treatment of TGF-ß measured by Western blot. (D) Western blot was conducted to test the expression EphB1 after transfection of Smad2 or knockdown of Smad2. (E) Expression of EphB1 after transfection of Smad2 or knockdown of Smad2 quantified by real-time PCR. (F) The putative Smad2/3 binding sites in EphB1 promoter. (G) ChIP assay in 293 cells to detect the binding of Smad2 to the promoter of EphB1. Mean values± SD of three independent experiments are shown on the right side. IgG indicates nonspecific Ab.

Fig 4: Ligand-independent EphB1 signaling promotes cancer cell migration and invasion. (A) EphB1 expression in NSCLC cell lines. (B) (C) Transwell assay was conducted to test the effect of EphB1 forced expression and (D) Ligand EphrinB2-Fc treatment on the migration and invasion of NSCLC cells. Number of cells were counted and shown in the column graph on the right of the corresponding pictures. Data are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01. (E) The proliferation of NSCLC was measured by CCK-8 or (F) Clonogenic assay after expression of EphB1 or knockdown of EphB1. Number of cell clones were counted and shown in the column graph on the right side. Data are mean ± SD of three independent experiments. ns: no significance.

Fig 5: EphB1 expression is correlated with poor patient survival in lung cancer. The expression of EphB1 was analyzed in patients with NSCLC. (A) EphB1 expression in NSCLC samples and non-cancer controls; the publicly accessible gene expression data of EphB1 was obtained from Gene Expression Ominibus (GEO) database (GSE10072, GSE19188, GSE7670) and The Cancer Genome Atlas (TCGA). (B) EphB1 expression in NSCLC patients with or without metastasis and normal controls or tumor samples; The publicly accessible gene expression data of EphB1 was obtained from Gene Expression Ominibus (GEO) database (GSE68465, GSE30219, GSE50081) and The Cancer Genome Atlas (TCGA). Data were analyzed with Student's t-test, p values were shown. (C) Kaplan-Meier overall survival curves according to EphB1 expression in patient cohorts in GEO datasets. The percentage of survival patients in high EphB1 and low EphB1 groups at different time points are presented. p values were shown. (D) EphB1 expression in lung cancer patient with or without metastasis was measured by immunohistochemical staining. Scale bar: Above=100 µm; Below=20 µm; Data were analyzed with Student's t-test, *p=0.0143.