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Figure 8. EV-PSMA^scFv ADAM9 silencing effects on androgen response and AR signaling in prostate cells. (A and D) Crystal violet viability assays in LNCaP (A) and CWR22Rv1 (D) cells following treatment with androgen-targeted therapies in combination with EV-PSMA^scFv loaded with siADAM9 variants (#1 and #2), non-targeting siRNA (siNC), or unloaded EVs. LNCaP cells were treated with ADT alone or combined ADT and Abi, whereas CWR22Rv1 cells were treated with abiraterone alone or combined ADT and abiraterone. Representative images and quantitative analysis of relative cell viability are shown; (B and E) RT-qPCR analysis of ADAM9, UBN2, and AKR1C3 expression in LNCaP (B) and CWR22Rv1 (E) cells following androgen-targeted treatment in combination with siADAM9-loaded EV-PSMA^scFv or control EV preparations. Relative gene expression was normalized to pre-treatment controls; (C and F) Immunoblot analysis of ADAM9-, AR signaling-, and cell cycle-associated proteins in LNCaP (C) and CWR22Rv1 (F) cells following treatment with androgen-targeted therapies and siADAM9-loaded EV-PSMA^scFv. Expression levels of ADAM9, UBN2, AKR1C3, AR-FL, AR-V7, p21, and β-actin were analyzed. Relative protein expression levels normalized to β-actin and referenced to the pre-treatment group are indicated below the blots. AR-V7 was undetectable in LNCaP cells and therefore was not quantified. Both AR-FL and AR-V7 were quantified in CWR22Rv1 cells. Blue values highlight notable expression changes observed in siADAM9-loaded EV-treated groups compared with siNC controls. Data were generated from three independent biological replicates (n = 3), each using a separately prepared EV batch, with triplicate analyses where applicable. Statistical analysis was performed using one-way (A and D) and two-way (B and E) ANOVA followed by Šídák’s multiple-comparisons test for the indicated comparisons. ns, not significant; ^*P < 0.05; ^**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; ADT: androgen deprivation therapy; Abi: abiraterone; RT-qPCR: reverse transcription quantitative polymerase chain reaction; UBN2: ubinuclein 2; AKR1C3: aldo-keto reductase family 1 member C3; AR: androgen receptor; AR-FL: full-length androgen receptor; AR-V7: androgen receptor splice variant 7; p21: cyclin-dependent kinase inhibitor 1A; SD: standard deviation; ANOVA: analysis of variance; β-actin: beta-actin; EV-PSMA^scFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments.