Figure 3.

Induction of DNA damage response pathways and apoptosis by adeno-associated virus (AAVs) in undifferentiated cell lines. (a) Induction of DDR, p53, and cell cycle arrest pathways by scAAV2 (5 × 10⁴ vg per cell) assessment by quantitative polymerase chain reaction. For these experiments, the viral vector with the highest effect, scAAV2, was used at a dose of 5 × 10⁴ vg per cell. The expression of key genes of the DDR, p53, and cell cycle arrest pathways was examined at the cDNA level. In particular, hES2C cells showed robust upregulation of BTG2 and p21 6 hours postinfection, whereas hiPS31.3C showed a significant increase in these two genes at 24 hours postinfection. Earlier time points for both cell lines showed no significant differences (data not shown). Some of the DDR and p53 pathway genes showed significant decrease in these time points (data not shown). Error bars depict ± standard error mean (SEM) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (b) Annexin V staining and flow cytometric analysis of hES2C and hiPS31.3C infected with scAAV2. hES2C at 6 and 9 hours postinfection (hpi) and hiPS31.3C 36 hpi with scAAV2 (1 × 10⁵ vg per cell) showed a significant increase, compared to no infection control, of Annexin V positive cells (early apoptosis), as well as of cells positive for both Annexin V and propidium iodide (PI) (late apoptosis). At later time points for hES2 (9 hpi) and at both time points for hiPS31.3 (24 hours and 36 hpi) cells were significantly positive for either Annexin V alone or Annexin V and PI. Error bars depict ± SEM (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).