Figure 3.

Enterovirus A71 (EV-A71) infection of differentiated C2BBe1 cells does not activate caspase 3 and is not affected by apoptosis inhibitors. (A) Differentiated and undifferentiated C2BBe1 were infected with EV-A71 at a multiplicity of infection (MOI) of 40. RD cells were infected with EV-A71 at an MOI of 0.2. Total protein was isolated from cells at different time points. Immunoblot assays were performed to detect the expression of procaspase 3, active caspase 3, and viral protein 3D. The expression of β-actin was used as an internal control. (B) Mock- and EV-A71-infected cells were fixed after 24 hours and allowed to react with mouse anti-EV-A71 3D and rabbit anti-active caspase 3 monoclonal antibodies (Abs), respectively. Phycoerythrin-conjugated antimouse immunoglobulin (Ig)G and fluorescein isothiocyanate-conjugated antirabbit IgG Abs were used for detection. (C) Differentiated C2BBe1 cells, undifferentiated C2BBe1, and RD cells were treated with Z-VAD-FMK (20 μM) or control medium and subsequently infected with EV-A71 at an MOI of 10 for undifferentiated and differentiated C2BBe1 cells and an MOI of 2 for RD cells. Total lysates (supernatants + infected cells) and supernatants were collected at different time points, and the viral titers were determined. The data are presented as the means + standard deviation (*, P < .05; **, P < .01). h p.i., hours postinfection; PFU, plaque-forming units.