Figure 3.

ICR4 and Poly(I:C) Induced Acute Cell Death, whereas ICR2 Induced Delayed Cell Death

(A–C) WM266-4 cells (2 × 10⁵ cells/well) were transfected for 4 hr with ICR2, ICR4, or poly(I:C) (1 μg/mL each) or transfection agent alone (Mock) in a 24-well plate. Cells and culture supernatants were harvested 4, 24, and 48 hr after transfection. (A and B) Cell death was determined using Annexin V and 7-AAD staining. % Cell death = (% Annexin V⁺/7-AAD⁻) + (% Annexin V⁻/7-AAD⁺) + (% Annexin V⁺/7-AAD⁺). (C) IFN-β production was determined by ELISA. (D and E) ICR2 but not ICR4 induced IFN-dependent cell death. WM266-4 cells (1 × 10⁴ cells/well) were transfected for 4 hr with ICR2, ICR4 (0.2 μg/mL each), or transfection agent alone (Mock) in a 96-well plate. Recombinant human IFN-β (100 ng/mL) was used as a positive control. Immediately after transfection, cells were cultured for 3 days in the presence or absence of B18R (1 μg/mL). Cytotoxicity was determined by Annexin V/7-AAD assay (D) and MTS assay (E). In (A), the data represent three individual experiments. In (D), the data represent two individual experiments. In (B), (C), and (E), the data are the mean of three experiments. Error bars show SD. *p < 0.05.