Figure 5. EphA2 drives tumor initiation and development partially through activation of STAT3 signaling.

(A) GSEA revealed that genes in the JAK/STAT3 pathway are highly enriched in EphA2 knockdown Huh7 cells.

(B) Cell lysate of EphA2 knockdown Huh7 and Hep3B cells were immunoblotted for p-STAT3 and total STAT3. GAPDH as a loading control.

(C) Liver of EphA2 knockout MET/CAT mice was extracted and immunohistochemically assessed for p-STAT3 expression comparing to control (PX330). Scale bar, 100 μm.

(D) Cell lysate of EphA2 knockdown Huh7 cells with overexpression of CA-AKT was immunoblotted for p-STAT3 and total STAT3.

(E) Western blot validated overexpression of STAT3 in the context of shRNA mediated EphA2 knockdown in Huh7 cells.

(F) Cell proliferation study was conducted using cells described in (C) using the Alamar blue assay. The ratio of proliferation was calculated by normalizing fluorescent intensity to day 0 (6 h after seeding of the cells) and subsequently measured at day 1, 2, and 4 days. Values are mean ± SD (n = 4).

(G) Left: representative picture of tumors extracted from NSG-A2 mice 28 days after subcutaneous injection with cells described in (E) and (F). n = 4 mice in each group. Right: primary tumor size was recorded every 2 days. Values are mean ± SEM.

(H) qPCR analysis of the indicated gene expression in HCC cells described in (F). Values are mean ± SD (n = 3).

(I) Left: representative picture of tumors extracted from NSG-A2 mice 28 days after subcutaneous injection with the indicated cells. n = 4 mice in each group. Right: primary tumor size was recorded every 2 days. Values are mean ± SEM.

Statistical significance was determined by one-ANOVA analysis with Tukey’s multiple comparisons test (F) and (G) and two-tailed Student’s t test (H). NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.