fig6
Figure 6. EV-PSMAscFv-mediated ADAM9 silencing effects on migration and EMT-associated remodeling in prostate cancer cells. (A and B) Wound healing assays in LNCaP (A) and CWR22Rv1 (B) cells following treatment with EV-PSMAscFv loaded with siADAM9 variants (#1 and #2), non-targeting siRNA (siNC), or unloaded EVs. Representative images were captured at the indicated time points, and wound closure percentages were quantified. Dashed lines indicate wound boundaries (scale bars: 500 μm); (C and D) Transwell migration assays in LNCaP (C) and CWR22Rv1 (D) cells following treatment with siADAM9-loaded EV-PSMAscFv or control EV preparations. Representative images of migrated cells are shown alongside quantitative analysis of cell migration based on the absorbance of solubilized crystal violet; (E and F) Immunoblot analysis of EMT- and ECM-associated proteins in LNCaP (E) and CWR22Rv1 (F) cells following treatment with siADAM9-loaded EV-PSMAscFv. Expression levels of E-cadherin, N-cadherin, MMP2, MMP9, TIMP3, and β-actin were evaluated. Relative protein expression levels normalized to β-actin are indicated below the blots. Representative images are shown. Data were obtained from three independent biological repeats, each using a separately prepared EV batch (n = 3 biological replicates; 3 independent EV preparations), with measurements performed in triplicate. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple-comparisons test. ns, not significant; **P < 0.01; ***P < 0.001. EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; siADAM9: ADAM9-targeting small interfering RNA; siNC: non-targeting small interfering RNA control; EMT: epithelial-mesenchymal transition; ECM: extracellular matrix; MMP2: matrix metalloproteinase 2; MMP9: matrix metalloproteinase 9; TIMP3: TIMP metallopeptidase inhibitor 3; SD: standard deviation; ANOVA: analysis of variance; β-actin: beta-actin; EV-PSMAscFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments.
