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. 2014 Jun 5;460:180–193. doi: 10.1016/j.virol.2014.04.040

Fig. 3.

Fig. 3

Pan-caspase inhibitor does not affect PEDV-induced apoptosis and PEDV infection. (A) FACS with dual Annexin V-PI cell labeling in the presence of Z-VAD-FMK. Vero cells were pretreated with vehicle or Z-VAD-FMK (100 μM) for 1 h and were mock-infected or infected with PEDV in the presence of vehicle or Z-VAD-FMK. Cells were harvested at the indicated time points, dually labeled with Annexin V and PI, and subjected to FACS analysis. The bottom graph represents the percentage of each quadrant. (B) Western blot analysis of caspase-3. At the indicated time, the cellular lysates were collected, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted using an antibody that recognizes caspase-3 (top panel) or PEDV N protein (third panel). The second panel was imaged after longer exposure to confirm the presence of activated caspase-3. The blot was also reacted with mouse MAb against the β-actin gene to verify equal protein loading (bottom panel). The figures are representative of three independent experiments. (C) PEDV replication in the presence of Z-VAD-FMK. Vero cells were treated with Z-VAD-FMK at the indicated concentrations for 1 h prior to infection and were infected with PEDV. PEDV-infected cells were further maintained for 48 h in the presence of vehicle or Z-VAD-FMK. PEDV-specific CPEs were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 200×(first panels). For immunostaining, infected cells were fixed at 48 hpi and incubated with MAb against the PEDV N protein, followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200×magnification. (D) Growth kinetics of PEDV in the presence of Z-VAD-FMK. At the indicated time points after infection, culture supernatants were harvested and viral titers were measured. Values are representative of the mean of three independent experiments and error bars represent standard deviations.