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. 2015 Nov 3;5:16185. doi: 10.1038/srep16185

Figure 4. Ailanthone induces caspase-dependent and caspase-independent apoptosis in Huh7 cells.

Figure 4

(A) Flow cytometric analysis of ailanthone-induced apoptosis in Huh7 cells using Annexin V-FITC/PI staining. Cells in the lower right quadrant (Annexin V+/PI−) represent early apoptotic cells, and those in the upper right quadrant (Annexin V+/PI+) represent late apoptotic cells. (B) Ailanthone activates caspase cleavage in a concentration- and time-dependent manner. Equal amounts of whole-cell extracts were separated by 10% SDS-PAGE, electrotransferred onto PVDF membranes, and analyzed by western blotting using the indicated antibodies against proteins related to caspase-dependent apoptosis. β-actin was used as a loading control. (C) Cells were pre-treated with the indicated concentration of z-VAD-fmk (20 μM) for 1 h prior to treatment. Cell viability was determined using MTT. The data are expressed as the mean ± SD of 3 independent experiments. ***P < 0.001 compared with the control group. (D) Huh7 cells were treated with various concentrations of ailanthone for 48 h. Western blotting was performed to detect the cytosolic levels of AIF and EndoG. β-actin and VDAC were used as the loading controls.