Fig. 3
Classification of cell death induced by BV-6 in KGN. Results of qRT-PCR, ATP-assay and Western Blot experiments performed to classify BV-6 induced cell death. (a) To test NF-κB pathway activation, qRT-PCR experiments of known target genes (BIRC4, BIRC2, BIRC3, IL8, TNFα and MCP-1) were performed. (a, upper graph) BIRC4, BIRC2, BIRC3 and IL8 levels were significantly increased after BV-6 stimulation (n = 5, geometric mean with 95% confidence interval, **p < 0.01, ***p < 0.001, ****p < 0.0001). (a, lower panels) MCP-1 and TNFα, both were absent in untreated samples but expressed in BV-6-treated samples (n = 5). Agarose gel shows a representative picture. The controls lacked template (H2O) or reverse transcriptase (−RT control). (b) ATP-assay was conducted in KGN that were stimulated with a combination of Z-VAD-FMK (1, 5, 20, 50, 200 μM) and BV-6 (EC50, 8 μM) for 24 h. Afterwards luminescence was normalized to cells solely treated with BV-6 (EC50, 8 μM). 1 μM and 5 μM Z-VAD-FMK had no effect on BV-6 induced cell death. 20 μM, 50 μM and 200 μM Z-VAD-FMK significantly reduced BV-6 induced cell death. (n = 4, mean and SEM, ***p < 0.001, ****p < 0.0001). (c,d) Western Blot analysis using specific α-clCASP3-, α-clPARP- and α-βActin -antibodies. KGN, treated with BV-6 (EC50, 8 μM) for 24 h were analyzed in comparison to controls. Western Blot revealed cleavage of (c) CASP3 and (d) PARP. The solvent treated controls lacked any apoptosis relevant signal but the loading control showed equal loading (n = 3, error bars indicate SEM)