Figure 2
From: Etoposide Induces Apoptosis in Activated Human Hepatic Stellate Cells via ER Stress
Effect of VP-16 on the induction of apoptosis in LX-2 cells.
(a) LX-2 cells were stained with JC-1 after incubation with VP-16 for 72 h to determine ΔΨm (magnification × 400). Undamaged mitochondria are hyperpolarized and stained red by the JC-1 dye. When mitochondria are damaged by VP-16, ΔΨm decreases and the JC-1 dye diffuses out of the mitochondria. Thus, the dye shows green fluorescence. (b) LX-2 cells were treated with various concentrations of VP-16 for 72 h. Apoptotic cells were analyzed using flow cytometry. The percentage of Annexin V-positive cells relative to untreated controls is indicated in a bar chart. (c) LX-2 cells were treated as described above. Western blotting was used to analyze the expression of PARP, cleaved PARP, XIAP, caspase-9, caspase-7, cytochrome c, caspase-3 and cleaved caspase-3. β-actin was used as a loading control. VP-16 increased the caspase-3 activities of LX-2 cells, which was assessed using a Caspase-3 Activity Assay kit. (d,e) LX-2 cells were pretreated with z-VAD-FMK (40 μM) for 1 h and then with 4 μM VP-16 for 72 h. (d) Annexin V-positive cells were examined by flow cytometry. PARP and cleaved PARP were measured by western blotting. β-actin was used as the loading control. (e) Cell viability was assessed using the CCK-8 assay. The results are expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
