Fig. 6

Effects of ROS on the intracellular distribution of rAAV2. A Localization of rAAV2-TAMRA in the early endosome when Hela cells were pretreated with H₂O₂ (100 nM) or without for 4 h and infected with rAAV2 at 2000 vg/cell at 37 °C. Early endosomes were measured using anti-EEA1 mAb after cell fixation at 1, 5, and 10 h post-infection. (Blue is the nucleus; Green is the early endosome; Red is rAAV2-TAMRA). B Localization of rAAV2-TAMRA in the late endosome when Hela cells were pretreated with H₂O₂ (100 nM) or without for 4 h and later infected with rAAV2 at 2000 vg/cell at 37 °C. Late endosomes were measured using anti-RAB7 mAb after cell fixation at 1, 5, and 10 h post-infection (Blue is the nucleus; Green is the late endosome; Red is rAAV2-TAMRA). C Percentage of rAAV co-localized with early endosome. D Percentage of rAAV co-localized with a late endosome. E WB assay on density-gradient fractions (#1–12; n = 12) collected in HeLa cells. The early endosome and late endosome markers EEA1 and RAB7 were detected by blotting. EEA1 is enriched in low-density fractions #10–12; RAB7 is enriched in high-density fractions #1–3. qPCR analysis of rAAV2 genomic in early endosomes (F and late endosomes G within 48 h of rAAV transduction with cells being pretreated with H₂O₂ for 4 h. The results are represented by means ± SD (n = 3). ^ns p > 0.05; *p < 0.05 vs. PBS; ** p < 0.01 ****p < 0.0001