Fig. 6.

Chelerythrine and staurosporine induce apoptosis and necroptosis in 661W cells. Cell death was examined by Annexin V/PI staining and flow cytometry. a Quantitation of apoptotic 661W cells following treatment with a range of concentrations of staurosporine (0–100 nM) for 4 h and 8 h in the presence of 5 % NHS or 5 % HI-NHS. b Example of the primary data for the 8 h, 100 nM staurosporine experiment. Apoptotic cells are Annexin V⁺ and locate to the top-left quadrant of the scatter plot whereas necroptotic cells are Annexin V and PI double positive and locate to the upper right quadrant. c Quantitation of necroptotic 661W cells from the same experiment as (a) and (b). d Necroptotic 661W cells were quantified as in (c) following treatment with 100 nM staurosporine for 8 h ± 10 µM AGK2. Necroptosis was significantly inhibited by AGK2. e Serum-starved and anti-CD59 treated 661W cells were incubated with 5 % NHS or 5 % HI-NHS for 1 h and then incubated with 0–20 µM chelerythrine for 4 or 8 h. Cells were then stained and subjected to flow cytometry as described above. The bar charts show the percentages of apoptotic cells. f Quantitation of necroptotic 661W cells from the same experiment as (e). g Representative scatter plots showing the results for cells treated with 20 µM chelerythrine for 8 h. (*P < 0.05, **P < 0.01, n = 3)