(A) Toxicity of inhibitors on HepG2/H-2Kb cells. HepG2/H-2Kb cells (2 × 10⁵ cells) were seeded on 12-well plates and incubated with different inhibitors. Cell viability was measured 24 hours later. (B) Effect of inhibitors on AAV2 transduction in HepG2/H-2Kb cells. HepG2/H-2Kb cells were incubated with inhibitors for 1 hour, then 1 × 10⁴ particles per cell of AAV2-OVA/luc vector were added to the culture for 24 hours. Cells were harvested for luciferase analysis. *P < 0.05 and **P < 0.01 when compared with AAV2/luc transduction without drug treatment. (C) Effect of inhibitors on antigen presentation. HepG2/H-2Kb cells were incubated with inhibitors for 1 hour and then transduced with 2 × 10¹⁰ AAV2-OVA/AAT vector for 24 hours. Cells were then fixed with 1% paraformaldehyde and washed, and OT-1 spleen cells were added to HepG2/H-2Kb cell culture medium. Activation of OT-1 spleen cells was determined by flow cytometry detecting CD8 and CD69 expression. The inhibition of antigen presentation was calculated as: [1-(number of activated OT-1 cells in inhibitor group with AAV2-OVA infection minus background group without AAV2-OVA infection)/(number of activated OT-1 cells in the group with AAV2-OVA infection – background group)] × 100%. *P < 0.05 and P < 0.01 when compared with the AAV2-OVA group without treatment. Data represent the average of 4 individual experiments and standard deviations. (D–H) Representative data from 1 experiment for acidification inhibitor (D), protease inhibitor (E), proteasome inhibitor (F), Golgi transporter inhibitor (G), and TPPII inhibitor (H).