Figure 2.

In vitro cytotoxic activity of Exo301

(A) Viability of HCT116 and SW48 cells 3 days after treatment with Exos or Exo301 at the indicated concentrations was assessed using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide) assay (n = 5). (B) Anti-CD63 antibody or IgG (for control) was added to the culture medium at the indicated concentrations together with Exo301 to inhibit Exo function, and the viability of HCT116 and SW48 cells was subsequently assessed using the XTT assay 3 days after treatment (n = 5). (C) Viability of HCT116 cells treated with EC or EC-Exo301 was assessed using the XTT assay 3 days after treatment (n = 5). (D) HCT116 cells were harvested 48 h after treatment with a 25% inhibitory concentration (IC₂₅) dose of OBP-301, Exo301, or EC-Exo301, or a dose of EC that was equivalent to that used in the EC-Exo301 isolation process. Whole-cell HCT116 lysates were then subjected to western blot analysis for PARP, p62, E1A, and β-actin. (E) HCT116 cells were treated with OBP-301, Exo301, or EC-Exo301 for 2 h and were harvested at the indicated time points after removing the treatments. The extracted DNA was subjected to qRT-PCR analysis of adenovirus E1A gene levels (n = 3). E1A copy numbers are described as fold change relative to time = 0 h. ∗p < 0.05, ∗∗p < 0.01. (F) HCT116 cells treated with an IC₂₅ dose of OBP-301, Exo301, or EC-Exo301 were harvested at the indicated post-treatment time points. Whole-cell HCT116 lysates were subjected to western blot analysis for PARP, p62, E1A, and β-actin.