Figure 2.

iNOS-induced cell apoptosis in FHM. (A) The protein overexpression of iNOS was detected by Western blot. FHM cells were transfected with 800 ng iNOS-3×Flag overexpression vector (pCMV-iNOS-3×Flag-CMV-eGFP, pCICE) in six-well plates. The control group was transfected with 800 ng empty vector (pCMV-CMV-eGFP, pCCE). Cell samples were collected after 24 h for Western blot. (B) iNOS overexpression induced cell apoptosis in FHM. FHM cells were transfected as (A) in glass-bottomed dishes. The samples were collected at 12 h, 24 h, 48 h, and 72 h and Incubated in 4% Paraformaldehyde and Hoechst 33342. The photos were imaged by fluorescence microscopy. There were no changes observed with the control group at 12 h. From 24 h to 72 h, the nuclei condensed and fragmented. The cells were broken and exploded into apoptosis bodies. As the white arrows show (scale 10 μm). (C) iNOS overexpression activated caspase-3 and caspase-9. FHM cells were transfected as (A) in the six-well plate. The samples were collected at 24 h, 48 h, and 72 h and the activity of caspase-3 and 9 were assayed by Caspase Activity Assay Kits. Data were analyzed as the mean ± SEM of the results from three independent experiments. *** p < 0.001.