CHX and Lexa Combination-Induced Apoptosis in HCC Figure 3. Lexa and CHX combination treatment induces apoptosis via ROS. A, Huh7 cells were treated with the combination of Lexa (1 mg/ml) and CHX (10 mg/ml). Nuclei were stained by Hoechst 33258 (right panel-blue, thin arrow marks the nucleus in non-apoptotic cells; thick arrow marks apoptotic condensed nuclei). Intracellular ROS level was assessed by DHED-based fluorescent staining (middle panel-red, thin arrow marks ROS in non-apoptotic cells, thick arrow marks ROS in apoptotic cells), and merged figure (left) to show that ROS levels in non-apoptotic cells are lower than that in apoptotic cells (white boxes marked areas were enlarged (Zoom in)). B, Huh7 cells were treated with DMSO (Con), Lexa (1 mg/ml), CHX (10 mg/ml), NAC (10 mM), a combination of Lexa (1 mg/ml) and CHX (10 mg/ml) in the absence or presence of NAC (10 mM). Intracellular ROS level was measured with DHED dye (red fluorescence). C, Huh7 cells were treated with various stimuli as indicated and apoptosis was assessed by DNA ladder. D, Huh7 cells treated with DMSO (Con), Lexa (1 mg/ml), CHX (10 mg/ml), NAC (10 mM), the combination of Lexa (1 mg/ml) and CHX (10 mg/ml) in the absence or the presence of NAC (10 mM) for 6 h. Then cells were stained with Hoechst 33258 to test apoptotic cell death (representative apoptotic cells are marked with arrows). E and F, Huh7 cells were treated with conditions as indicated. Apoptosis was evaluated as in Fig. 1C and 1D. Data represent the mean values of three independent experiments (**p,0.05). doi:10.1371/journal.pone.0016966.g003 PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e16966