fig3

Figure 3. PSMA expression determines binding efficiency of EV-PSMA^scFv. (A) Flow cytometric analysis of surface PSMA expression in LNCaP cells following siRNA-mediated PSMA knockdown (siPSMA). Mock-transfected and siNC controls are shown. Representative histograms and quantification of MFI are presented; (B) Flow cytometric analysis of PSMA surface expression in WT and PSMA-OE PC3 cells. Representative histograms and quantification of MFI are shown; (C and D) Flow cytometric analysis of EV binding in mock-, siNC-, and siPSMA-treated LNCaP cells following exposure to labeled EV-PSMA^scFv. Representative dot plots (C) and quantitative analysis of MFI (D) are shown; (E and F) Flow cytometric analysis of EV binding in WT and PSMA-OE PC3 cells following exposure to labeled EV-PSMA^scFv. Representative dot plots (E) and quantitative analysis of MFI (F) are shown. Data were obtained from three independent biological repeats (n = 3), with each repeat using a separately prepared EV batch and analyzed in triplicate. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple-comparisons test (A and B), one-way ANOVA followed by Tukey’s multiple-comparisons test (D), and Student’s t-test (F). ns; ^**P < 0.01; ^***P < 0.001. EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; siRNA: small interfering RNA; siPSMA: PSMA-targeting small interfering RNA; siNC: non-targeting small interfering RNA control; WT: wild-type; PSMA-OE: PSMA-overexpressing; MFI: mean fluorescence intensity; ANOVA: analysis of variance; ns: not significant; EV-PSMA^scFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments; FITC-A: fluorescein isothiocyanate area; PE-A: phycoerythrin area.