Fig. 7: Functional validation identifies SHB as a mediator of EphA2-induced Erk-pathway inhibition and afadin as an EphA2-regulated phosphoprotein that mediates cell invasion in PC-3 prostate cancer cells.

a Western blot analysis of the effect of transient SHB siRNA knockdown on Akt and Erk pathway inhibition in response to EphA2 activation. Cell lines were incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (efnA1-Fc) for 20 min. A representative western botting image is shown. β-actin is the loading control. Relative phosphorylated protein levels of phospho-EphA, phospho-Erk and phospho-AKT were quantified and are shown as bar charts. Mean ± SE (n = 3 biological replicates shown as individual data points). SHB Knockdown was confirmed by quantitative QPCR and relative mRNA expression levels determined using the 2^−ΔΔCt method with β-actin as the housekeeping gene and the Lipofectamine control as the reference sample. Mean ± SE (n = 3 biological replicates). Knockdown of SHB diminished the ligand-induced EphA2-mediated Erk1/2 pathway inhibition, while the inhibitory effect on the Akt pathway remained intact. b Representative confocal immunofluorescence images of afadin (AFDN, red) and EphA2 (green) cellular localization in response to EphA2 stimulation with clustered ephrin-A1-Fc (efnA1-Fc). EphA2 activation regulates the intracellular localization of afadin. Cell nuclei are labeled with DAPI (blue). Scale bars are 20 µm. c Transient knockdown of afadin (AFDN) with two different siRNA sequences was confirmed by western blot analysis. β-actin is the loading control. Bar chart shows knockdown efficiency as determined by densitometry of western blotting results. Mean ± SE (n = 3 biological replicates). ***p value < 0.001, ns. not significant (One-way ANOVA with post-hoc Šidák test for multiple pairwise comparisons). d Transwell invasion assay comparing invasion of afadin (AFDN) knockdown cells compared to untransfected and negative control siRNA-transfected PC-3 cells. Mean ± SE (n = 5 biological replicates). *p value < 0.05, ns. not significant (One-way ANOVA with post-hoc Šidák test for multiple pairwise comparisons).