EphB4 promotes melanoma cell proliferation and inhibits apoptosis in vitro and promotes melanoma tumor growth in vivo. (A) EphB4 expression levels in 3 SW1, C19 and C19-EphB4 clones (data for clone #1 are shown in Figure S2). Lysates were probed by immunoblotting for EphB4, and GAPDH as a loading control. (B) Cell proliferation assessed by measuring BrdU incorporation. The histograms show average percentages of BrdU-positive cells 2 hours following BrdU application ± SEM (n ~1,000 cells per group from 3 independent experiments; ***p < 0.001 compared with the C19 cells by one-way ANOVA). (C) Apoptosis assessed by Hoechst 33342 staining. The histograms show average percentages of Hoechst-positive cells ± SEM (n ~2,250 cells per group from 3 independent experiments; ***p < 0.001 compared with the C19 cells by one-way ANOVA). (D) Photographs of representative 7.5 week-old tumors derived from a mixture of 3 clones (#2, #3 and #4) of SW1, C19 or C19-EphB4 cells. The growth curves show average tumor volumes ± SEM measured at the indicated times in groups of 6 mice. **p < 0.01 compared with the C19 group using repeated measures two-way ANOVA. The histograms show average tumor weights and body weights ± SEM measured at 7.5 weeks (n = 6 tumors per group; **p < 0.01 compared with the C19 group by one-way ANOVA).

EphB4 upregulates Erk and Akt phosphorylation and Bcl-2 expression, and promotes blood vessel enlargement in melanoma tumors. (A) EphB4 immunoprecipitates from tumors grown for 7.5 weeks were probed for phosphotyrosine (pTyr) and reprobed for EphB4. The histogram shows average levels of phosphorylated EphB4 quantified by densitometry and normalized to total EphB4 ± SEM (n = 4 tumors per group; ***p < 0.001 compared with the C19 tumors by one-way ANOVA). (B, C, D) Tumor lysates were probed for phosphoErk1/2 Thr202/Tyr204 and total Erk1/2, phosphoAkt S473 and total Akt, Bcl-2 and GAPDH. The histograms show the average levels of pErk, pAkt or Bcl-2 quantified by densitometry and normalized to total Erk, total Akt or GAPDH ± SEM (n = 4 tumors per group; *p < 0.05; **p < 0.01; ***p < 0.001 compared with the C19 tumors by one-way ANOVA). The lanes in each blot are from the same gel and the white space indicates that an irrelevant lane between the SW1 and C19 lanes was removed. (E) Fluorescent images of EGFP-positive melanoma cells (green) and blood vessels stained with anti-CD31 antibody (red) in frozen sections from tumors collected at 7.5 weeks. The histograms show average areas occupied by blood vessels and number of blood vessels per image (image area 180 mm²). Scale bar, 2 µm. The histograms show averages ± SEM (n = 16 sections from 3–4 mice corresponding to ~1,000 blood vessels per group; *p < 0.05; **p < 0.01; ***p < 0.001 compared to C19 tumors by one-way ANOVA).