Fig. 2

Cell death induced by shikonin in A549 cells occurs by necroptosis. a Cells were pretreated for 2 h in the presence or absence of Nec-1 (100 μM), Z-VAD (20 μM), Z-DEVD (20 µM), Z-IETD (10 µM), or Z-LEHD (20 µM) and then incubated with shikonin (3 or 6 µM) for 3 or 6 h. Viability was then measured by the MTT assay. Values are represented as the mean ± SD of three independent experiments. *p < 0.01 compared with the control, **p < 0.05 compared with the shikonin group. b Cells were pretreated for 2 h in the presence or absence of Nec-1 (100 µM) or Z-VAD (20 µM) and incubated with shikonin for 3 h. Necroptosis was evaluated by annexin V/PI staining. c Values are presented as mean ± SD of three independent experiments. *p < 0.05 compared to control. d Cells were pretreated for 2 h in the presence or absence of Nec-1 (100 µM) or Z-VAD (20 µM) and then treated with shikonin (3 or 6 μM) for 3 h. The cells were then lysed, and the cell lysate was subjected to 10 and 15% SDS-PAGE to measure the expression of the indicated proteins. The levels of RIP1, cleaved PARP, and cleaved caspase 3 were measured by western blotting. Data are representative of two independent experiments