Annexin V-positive/PI-negative cells are in early stages of apoptosis and double positive cells are in late apoptosis INCB024360 cell line (B) *P < 0.05 vs Control,#P < 0.01 vs Control,▲P < 0.05 vs 10 μg/ml NCTD,※P < 0.05 vs 20 μg/ml NCTD Generation of ROS in HepG2 cells treated with NCTD ROS generation was analyzed by flow cytometry. Cells were treatment with various concentrations of NCTD (10, 20, 40 μg/ml) for 24 h, and then DCF fluorescence was recorded as a measure of intracellular

ROS. As shown in Figure 4A, the treatment of HepG2 cells with NCTD resulted in a dose-dependent increase in ROS generation. As shown in Figure 4B, the result demonstrated that the NAC pretreated cells reduced levels of FL-1 fluorescence of DCF. Figure 4 Effect of NCTD on ROS generation in HepG2 cells. (A) Cells were treated with NCTD for 6 h, followed by staining with DCHF-DA (100 μM) for an additional 30 min. NAC(10 mM) was added 1 h prior to Wnt inhibitor the treatment with 20 μg/ml NCTD for 6 h.Cells treated with NCTD showed a dose-dependent increase in ROS generation. The horizontal axis represents DCFH-DA fluorescence and the vertical axis represents cell count. (B) *P < 0.01 vs Control,§P < 0.05 vs 10 μg/ml NCTD,▲P < 0.05 vs 20 μg/ml NCTD,#P < 0.01 vs 20 μg/ml NCTD Mitochondria Membrane Potential (Δφm) Determination Disruption of mitochondrial integrity is

one of the early events leading to apoptosis. To assess whether NCTD affects the function of mitochondria, potential changes in mitochondrial membrane were analyzed by employing a mitochondria fluorescent dye, JC-1. As shown in Figure 5, exposure to NCTD for 24 h resulted in a significant decrease in the ratio between red and green fluorescence by approximately 33.83 ± 1.53%, 45.23 ± 0.78%, and 56.6 ± 0.85% at 10, 20 and 40 μg/ml, respectively. This suggests that treatment with various concentrations of NCTD (10, 20, 40 μg/ml) for 24 h resulted in significant decreases of Δφm. The results imply that NCTD induces Δφm dissipation SPTLC1 in a concentration-dependent manner. Figure 5 NCTD-Induced Δφm Depolarization in HepG2 Cells. (A) Cells were treated

Sepantronium research buy without or with NCTD for 24 h at the concentrations indicated. Change in Δφm was determined by flow cytometric analysis with JC-1. (B) *P < 0.01 vs Control,§P < 0.01 vs 10 μg/ml NCTD,▲P < 0.01 vs 20 μg/ml NCTD. Cytochrome c Release from Mitochondria to Cytosol Cytochrome c release from mitochondria is a critical step in the apoptotic cascade since this activates downstream caspases. To investigate the release of cytochrome c in NCTD-treated HepG2 cells, we conducted western blotting in both the cytosolic and mitochondrial fractions. The results demonstrate a concentration-dependent increase in the cytosolic cytochrome c after treatment with NCTD. Simultaneously, there was a decrease in cytochrome c in the mitochondrial fraction (Figure 6A). Figure 6 Effect of NCTD on Expression of Cyto-C, Bax/Bcl-2/Bid, c aspase-3/-8/-9 and PARP proteins in HepG2 Cells.