Figure 7.

CsA treatment diminishes SADS-CoV-induced apoptosis and suppresses SADS-CoV propagation. (A) CsA treatment does not affect cell viability. Vero E6 and IPI-2I cells were treated with the carrier control DMSO or CsA at different concentrations for 36 h. Cell cytotoxicity was analyzed by CCK-8 kit as described in Materials and Methods. (B) FACS with dual Annexin V-PI cell labelling in the presence of CsA. Vero E6 cells were pretreated with DMSO or CsA (10 μM) for 1 h and mock-infected or infected with SADS-CoV in the presence of DMSO or CsA. Cells were harvested at the indicated time points, dual labelled with Annexin V and PI, and analyzed by FACS. The graph on the right represents the percentage of each quadrant. (C) DNA fragmentation analysis in the presence of CsA. Vero E6 cells were pre-incubated with Z-VAD-FMK (100 μM) or CsA (10 μM) for 1 h and infected with mock virus or SADS-CoV. Nucleosomal DNA fragmentation of the cells was analyzed by agarose gel electrophoresis. Lane M, 2-kb DNA molecular weight marker; lane 1, mock-infected and non-treated; lane 2, only SADS-CoV-infected; lane 3, SADS-CoV-infected and Z-VAD-FMK-treated; lane 4, SADS-CoV-infected and CsA-treated. (D) SADS-CoV infection in the presence of CsA. Vero E6 cells were treated with DMSO or CsA at the indicated concentrations for 1 h prior to their infection with SADS-CoV. SADS-CoV-infected cells were further maintained for 36 h in the presence of DMSO or CsA. For immunostaining, infected cells were fixed at 36 hpi and stained with an anti- SADS-CoV N protein antibody, followed by incubation with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody. The cells were counterstained with DAPI and examined under an inverted fluorescence microscope. The percentage of SADS-CoV infected cells per view from three independent experiments is expressed as the mean ± SD. (E and F) Viral N protein expression and PARP cleavage in the presence of CsA. Vero E6 and IPI-2I cells were treated with DMSO or CsA at the indicated concentrations for 1 h prior to infection with SADS-CoV. SADS-CoV infected cells were maintained for 36 h in the presence of DMSO or CsA. At 36 hpi, cellular lysates were examined by western blot with antibodies against SADS-CoV N protein and PARP. The blot was also reacted with a mouse mAb against GAPDH to verify equal protein loading. Densitometric data for N/GAPDH and cleaved PARP/GAPDH from three independent experiments are expressed as the mean ± SD. (G and H) CsA treatment suppresses SADS-CoV replication. Treatment and infection conditions were as described in panel E and F, and the viral titers in the supernatants collected at 36 hpi were determined by the Spearman-Kärber method. Error bars represent the standard errors of the means from three independent experiments.