Fig. 3. FACScan analysis of apoptotic melanoma cells (melanoma cell line 530) after exposure to hypoxia and in vitro luciferase assays of c-Jun transcriptional activity. A and B, apoptotic cells were identified by staining with biotin-conjugated annexin V. Melanoma cells under normoxic conditions and hypoxia (12 h/24 h) were incubated in 200 µl of annexin buffer containing 4 µl of annexin V-biotin for 30 min at 4°C. After that, cells were incubated with streptavidin cytochrome c and analyzed by flow cytometry (FACScan). Cells were transiently transfected with the wild-type or dominant negative interfering kinases of SAPKß54 or SEK1 or exposed to the chemical inhibitors PD98058 or SB203580. Representative FACScan dot plots of one experiment are shown in A. Diagrams in B show the mean ± SD of the percentage of apoptotic melanoma cells from four independent experiments. Asterisks (*, **) indicate the statistical significance of reduced apoptosis after transfection with the dominant negative interfering kinases compared to the wild-type kinases (P 0.05). C, in vitro luciferase assays of c-Jun transcriptional activity after transfection of melanoma cells with wild-type and dominant negative interfering kinases (SAPKß54, SAPK KR, SEK, and SEKKR), respectively. Asterisks (*, **) indicate the statistical significance of reduced c-Jun-dependent transcriptional activity after transfection with the dominant negative interfering kinases.