Figure 3.

Intracellular ROS is involved in cellular apoptosis induced by SFN. (A) Cells were exposed to the indicated concentrations of SFN for 24 h. The level of intracellular ROS was assessed using DCFH-DA staining and observed by fluorescence microscope. As a positive control, cells were treated with pyocyanin (200 µM). Bar: 20 µm; (B) cells were pre-incubated with or without NAC (3 mM) for 1 h followed by the addition of SFN (40 µM) for a further 24 h. Cell viability was measured by CCK-8 assay; (C) cells were treated with SFN (40 µM) in the absence or presence of NAC. The apoptosis was determined by Annexin V/PI staining and analyzed by flow cytometry. Cells in Q2 quarters (Annexin V positive/PI negtive) represented early apoptotic cells. The double-positive cells in Q3 quarters represented late apoptotic cells; (D) the percentages of late apoptotic cells relative to the total population were quantified; (E) cells were treated with SFN (40 µM) for 24 h after pre-incubation with or without NAC (3 mM). The cell lysates were subjected to Western blot using anti-cleaved caspase3, anti-Bcl-2 and anti-Bax, antibodies; and (F) the bands corresponding to each protein were quantified and normalized relative to band intensities for control group. Data are represented as means ± SD. ** p < 0.01, and *** p < 0.001 indicates difference between SFN alone versus co-treatment with NAC.